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Role of Receptor Tyrosine Kinase Regulator Sprouty in Ovarian Cancer Cells

by Wai Kin So BSc, The Chinese University of Hong Kong, 2002 MPhil, The Chinese University of Hong Kong, 2004

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

in

The Faculty of Graduate Studies (Reproductive and Developmental Sciences)

THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April, 2012 © Wai Kin So 2012

Abstract

Aberrant epidermal growth factor receptor (EGFR) activity contributes to the development of epithelial ovarian cancer (EOC), a common and lethal female malignancy. Elucidating the regulation of EGFR function will improve treatments for EOC and the survival of patients.

This study aims to elucidate the role of Sprouty (SPRY) proteins, which are EGFR regulators, in EOC. The investigation began with demonstrating the downregulation of mRNA levels of two SPRY members, SPRY2 and SPRY4, in EOC tissues and/or cell lines. Deletion of the SPRY2 gene was found to cause reduced SPRY2 mRNA. Loss of the SPRY2 gene and thus its expression are particularly common in high-grade serous tumors, suggesting that SPRY2 deficiency may be involved in the pathogenesis of this prevailing subtype of EOC. The regulatory mechanisms of SPRY level are incompletely understood. The EGFR ligand EGF strongly upregulates SPRY4 protein level primarily through the ERK pathway. In addition, the PI3K/AKT pathway and hypoxia-inducible factor-1 (HIF-1α) have been shown to be involved in SPRY4 regulation, allowing the possibility that SPRY4 is regulated by micro-environmental (hypoxia) and genetic (PI3K mutation) abnormalities.

Functionally, SPRY2 and SPRY4 counteract various aspects of EGFR activity and generally have tumor suppressor functions. First, in contrast to the EGFR, SPRY2 and SPRY4 prevent loss of cell adhesion by E-cadherin and therefore suppress cancer cell invasion. Second, SPRY4 inhibits PI3K/AKT signalling activated by EGF, as AKT

activation is enhanced in the absence of SPRY4. Finally, the HIF-1α oncogene has been identified as a novel SPRY4 target. In ovarian cancer cell lines, SPRY4 suppresses the basal and EGF-stimulated expression of HIF-1α. The negative effects of SPRY4 on HIF- 1α are also reflected by modulation of HIF-1 activity and target gene expression. SPRY4 has also been shown to destabilise HIF-1α protein, independent of the classic HIF-1α degradation pathway. The current study investigated the expression, regulation and function of SPRY in ovarian cancer. Understanding the tumor suppressor role of SPRY will not only enhance our knowledge about the pathophysiology of ovarian cancer but also identifies a possible therapeutic intervention against this lethal malignancy.

Preface

1. A version of Chapter 2 has been submitted and is under revision. Wai-Kin So, Alice S. T. Wong, David G. Huntsman, C. Blake Gilks, and Peter C. K. Leung. Genetic Inactivation of Sprouty2 Promotes Epidermal Growth Factor-induced E-cadherin Downregulation and Invasion in Ovarian Cancer Cells.

Contributions

David G. Huntsman, C. Blake Gilks and I participated in design and performance of the study. I drafted the manuscript. Peter C. K. Leung, Alice S. T. Wong, David G. Huntsman and C. Blake Gilks read, critically revised and approved the manuscript.

Ethics Approval

Approvals for the study were obtained from the University of British Columbia Research Ethics Board (#H04-60102, #H02-61375 and #H03-70606) and written informed consents from all participants involved in the study were obtained.

2. A manuscript based on a version of Chapter 3 has been under preparation. Wai-Kin So, Man-Tat Lau, and Peter C. K. Leung. An Amphiregulin and Sprouty4 Loop Regulates Ovarian Cancer Cell Invasiveness Via and E-cadherin-dependent Mechanism.

Contributions

I participated in design of the study, Man-Tat Lau and I performed experiments. I drafted the manuscript.

iv

Presentation

  • 5 th International Epithelial-Mesenchymal Transition Meeting, Singapore, Oct. 2011

Poster presentation on An Amphiregulin and Sprouty4 Loop Regulates Ovarian Cancer Cell Invasiveness Via and E-cadherin-dependent Mechanism. Wai-Kin So and Peter C. K. Leung.

Table of Contents

Abstract

ii!

iv!

Table of Contents

vi!

List of Tables

ix!

List of Figures

x!

List of Abbreviations

xii!

Acknowledgements

xvi!

Chapter 1 Introduction

1!

  • 1.1 Ovarian cancer

..........................................................................................................

1!

  • 1.2 Tumorigenesis of EOCs

1!

  • 1.3 Classification of EOCs

..............................................................................................

3!

  • 1.3.1 High-grade serous carcinoma (HGSC)

3!

  • 1.3.2 Low-grade serous carcinoma (LGSC)

5!

  • 1.3.3 Endometrioid carcinoma (EC)

6!

  • 1.3.4 Clear cell carcinoma (CCC)

7!

  • 1.3.5 Mucinous tumors

7!

  • 1.4 Epidermal growth factor (EGF) and the EGF receptor (EGFR)

8!

  • 1.4.1 EGFR structure and signaling

8!

  • 1.4.2 EGFR expression and mutations in ovarian cancer

...........................................

9!

  • 1.4.3 EGFR

ligands ...................................................................................................

11!

  • 1.4.4 Functions of the EGFR and it ligands in ovarian cancer

13!

  • 1.4.5 Clinical activities of EGFR-targeted

15!

  • 1.5 Sprouty (SPRY)

16!

  • 1.5.1 SPRY

16!

  • 1.5.2 SPRY functions and mechanisms

17!

  • 1.5.3 Regulation of SPRY activity

18!

  • 1.5.4 SPRY expressions in cancer

............................................................................

19!

  • 1.5.5 SPRY as tumor suppressor genes

19!

  • 1.6 Hypoxia-inducible factor-1 alpha (HIF-1α)

............................................................

19!

  • 1.6.1 HIF-1α

structure ...............................................................................................

20!

  • 1.6.2 HIF-1α regulation

............................................................................................

21!

  • 1.6.2.1 Hypoxia .....................................................................................................

21!

  • 1.6.2.2 Regulation of HIF-1α in normoxia

22!

VHL mutations

  • 1.6.2.2.1 ...................................................................................

22!

AKT and PI3K

  • 1.6.2.2.2 ...................................................................................

23!

  • 1.6.2.2.3 Glycogen synthase kinase 3β (GSK3β)

25!

  • 1.6.2.2.4 Heat shock protein 90 (HSP90)

26!

  • 1.6.2.2.5 Hormonal regulation

27!

  • 1.6.3 HIF-1α expressions in cancer

27!

  • 1.6.4 HIF-1α functions in cancer

28!

1.6.4.1

Angiogenesis .............................................................................................

28!

1.6.4.2

Metastasis ..................................................................................................

29!

  • 1.7 Hypothesis and

30!

  • 2.1 Introduction .............................................................................................................

37!

  • 2.2 Materials and methods

............................................................................................

39!

  • 2.2.1 The Human Exonic Evidence-Based Oligonucleotide microarray (HEEBO) . 39!

  • 2.2.2 Molecular inversion probe (MIP) copy number analysis

39!

  • 2.2.3 The Cancer Genome Atlas (TCGA)

40!

Real-time PCR

  • 2.2.5 .................................................................................................

41!

  • 2.2.6 Antibodies ........................................................................................................

41!

Invasion assay

  • 2.2.7 ..................................................................................................

42!

  • 2.2.8 Statistical analysis ............................................................................................

42!

  • 2.3 Results .....................................................................................................................

43!

  • 2.3.1 Levels of SPRY mRNA in ovarian tumors of different pathological subtypes 43!

  • 2.3.2 Levels of SPRY mRNA in immortalised ovarian surface epithelium (IOSE) and

EOC-derived cell lines

44!

  • 2.3.3 A deletion event in the proximity of the SPRY2

44!

  • 2.3.4 SPRY2 deletion may lead to reduced SPRY2 mRNA level

45!

  • 2.3.5 SPRY2 reversed EGF-suppressed E-cadherin protein expression and

antagonized EGF-induced cell invasion

46!

  • 2.3.6 SPRY2 and E-cadherin proteins displayed a positive correlation in human

ovarian cancer cell lines and tumors

47!

  • 2.4 Discussion

47!

Chapter 3 An amphiregulin and Sprouty4 loop regulates ovarian cancer cell invasiveness

via an E-cadherin-dependent mechanism

.........................................................................

60!

  • 3.1 Introduction .............................................................................................................

60!

  • 3.2 Materials and methods

............................................................................................

62!

  • 3.2.1 Cell culture and reagents

62!

Transfection

  • 3.2.2 .....................................................................................................

63!

Real-time PCR

  • 3.2.3 .................................................................................................

63!

  • 3.2.4 Western blot analysis

64!

Invasion assay

  • 3.2.5 ..................................................................................................

64!

  • 3.2.6 Statistical analysis ............................................................................................

65!

  • 3.3 Results .....................................................................................................................

65!

  • 3.3.1 AREG promoted invasion of ovarian cancer cells

...........................................

65!

  • 3.3.2 AREG reduced E-cadherin levels, and E-cadherin overexpression blocks

AREG-induced invasion

...........................................................................................

66!

  • 3.3.3 AREG suppressed E-cadherin level and promotes cell invasion via the EGFR

 

66!

  • 3.3.4 AREG induced Slug

67!

  • 3.3.5 The MAPK/ERK and PI3K/AKT pathways mediated the effects of AREG on

SLUG mRNA and E-cadherin levels and cell invasion

67!

  • 3.3.6 AREG induced SPRY4 expression

..................................................................

67!

  • 3.3.7 SPRY4 knockdown enhanced AREG-induced E-cadherin suppression and

invasion

68!

  • 3.4 Discussion

68!

Chapter 4 Sprouty4 feedback regulates epidermal growth factor/AKT/hypoxia-inducible

factor-1 alpha axis in ovarian cancer cells

82!

4.1

Introduction .............................................................................................................

82!

4.2

Materials and methods

84!

4.2.1

Cell culture and reagents

84!

4.2.2

Transfection

.....................................................................................................

84!

4.2.3

Real-time PCR

.................................................................................................

85!

4.2.4

Western blot analysis

85!

4.2.5

Luciferase assay

86!

4.2.6

Statistical analyses

86!

4.3

Results .....................................................................................................................

87!

4.3.1

EGF increased SPRY4 in ovarian cancer cells

87!

4.3.2

The MEK/ERK and PI3K/AKT pathways mediated the effects of EGF on

SPRY4 levels

............................................................................................................

87!

4.3.3

EGF induced HIF-1α via the PI3K/AKT pathway

..........................................

88!

4.3.4

HIF-1α plays a minor role in EGF-induced SPRY4 level

88!

4.3.5

SPRY4 overexpression reversed EGF-induced HIF-1α levels and HIF-1

 

89!

4.3.6

SPRY4 knockdown enhanced the effect of EGF on HIF-1α

89!

4.3.7

AKT pathway mediated HIF-1α regulation by EGF and SPRY4

90!

4.4

Discussion

90!

5.1

Introduction

103!

5.2

Materials and methods

..........................................................................................

105!

5.2.1

Cell culture and reagents

105!

5.2.2

Transfection

105!

5.2.3

Real-time PCR

106!

5.2.4

Western blot analysis

106!

5.2.5

Statistical analysis

107!

5.3

Results

107!

5.3.1

SPRY4 negatively regulated HIF-1α expression levels in ovarian cancer cells

 

107!

5.3.2

SPRY4 negatively regulated HIF-1 activity

108!

5.3.3

levels

109!

5.3.4

HIF-1α modulation by SPRY4 was independent of PHD activity

109!

5.4

Discussion

110!

References

129!

List of Tables

Table 2.1 Comparison between mean SPRY2 mRNA levels of various histopathological types using Student’s t test ..……….…………………………….…………………...…..51

Table 2.2 MIP analysis of loss of the markers flanking the SPRY2 and SPRY4 loci in ovarian tumors……………………………………………………………………… ….52 ..

ix

List of Figures

Figure 1.1 The chart illustrates the pathogenesis of epithelial ovarian cancers…..……..33

Figure 1.2 The structure and signaling of EGFR.…………..……...…………………….34

Figure 1.3 A schematic diagram of human SPRY depicting its structure, its interacting

partners and the corresponding functional consequences. ………………………………35

Figure 1.4 The diagram illustrates the regulation of HIF-1α. ……………………..……36

Figure 2.1 The box plot displays the mean SPRY2 mRNA levels in serous (high-grade,

low-grade or borderline), endometrioid (carcinoma or borderline tumor), clear cell

ovarian tumors and normal Fallopian tube. (HEEBO array data)……………………….54

Figure 2.2 Comparison of SPRY2 and SPRY4 mRNA levels in immortalised OSE (IOSE)

and ovarian cancer cell lines by real-time PCR

...

…………………………………… …55

..

Figure 2.3 A schematic representation of the MIP copy number assay results of 28 high-

grade serous carcinomas ...………………………………………………………...….….56

Figure 2.4 The effect of SPRY2 on EGF-induced E-cadherin suppression and cell

invasion.…………………

...

……………………………………………………………..57

Figure 2.5 The correlation between SPRY2 and E-cadherin protein expression in ovarian

cancers ..…………………………………………………..…………………………..….59

Figure 3.1 AREG promoted ovarian cancer cells invasion.….…………………… … 73

..

...

Figure 3.2 AREG reduced E-cadherin levels and E-cadherin overexpression blocked

AREG-induced invasion. ……………… ………………………………………………74 ..

Figure 3.3 AREG suppressed E-cadherin level and promoted cell invasion via the EGFR.

………………………………………………………………………………….……… 75

..

Figure 3.4 AREG induced SLUG mRNA ……………………………….…………… 76 ....

Figure 3.5 The MAPK/ERK and PI3K/AKT pathways mediated the effects of AREG on

Slug and E-cadherin level and cell invasion. ………………………………………… 77 ....

Figure 3.6 AREG induced SPRY4………………………………………………… … 79

..

..

Figure 3.7 SPRY4 knockdown enhanced AREG-induced E-cadherin suppression and

invasion ..……………………………………………...…………..……………..……….80

Figure 4.1 EGF induced SPRY4 level in ovarian cancer cells ...…………………..…….94

Figure 4.2 The MEK/ERK and PI3K/AKT pathways mediated the effects of EGF on

SPRY4 levels..………………………………………………………...…………………95

Figure

4.3

EGF

induced

HIF-1α

and

HIF-1

activity

via

the

PI3K/AKT

pathway.………………………………………………………………………………….96

Figure 4.4 HIF-1α plays a minor role in EGF-induced SPRY4 level ..…...……………..97

Figure 4.5 SPRY4 overexpression reversed EGF-induced HIF-1α expression and HIF-1

activity.……………………………………………………………………….……… …99 ..

Figure 4.6 SPRY4 knockdown enhanced EGF effect on HIF-1α...…………..…..........101

Figure 4.7 The PI3K/AKT pathway mediated HIF-1α regulation by EGF and

SPRY4……………………………………………………………………………… 102 .....

Figure 5.1 SPRY4 negatively regulated HIF-1α levels in ovarian cancer cells.

………………………………………………………………………………… ….… 113

...

..

Figure 5.2 SPRY4 negatively regulated HIF-1 activity. ……………………….………114

Figure 5.3 SPRY4 regulated HIF-1α protein half-life without affecting Hif-1α mRNA

levels. …… ………………………………

... ...

…….……………………….… 115 ..............

Figure 5.4 HIF-1α modulation by SPRY4 acts independently of PHD activity.

………………………………………………………………………………………… 117 ..

Figure 6.1 The diagram summarizes the findings. ……………………………………..128

List of Abbreviations

AREG

Amphiregulin

BRCA1

Breast cancer 1, early onset

BTC

Betacellulin

CCC

Clear cell carcinoma

CTNNB

Cadherin-associated protein beta

DM

Double mutant

DMSO

Dimethyl sulfoxide

EC

Endometrioid carcinoma

ECD

Extracellular domain

ECM

Extracellular matrix

EGF

Epidermal growth factor

EMT

Epithelial-mesenchymal transition

EOC

Epithelial ovarian cancer

EPI

Epiregulin

ERBB

Erythroblastic leukemia viral oncogene homolog

ERK

Extracellular signal regulated protein kinase

FBS

Fetal bovine serum

FGF

Fibroblast growth factor

GAPDH

Gylceraldehyde-3-phosphate dehydrogenase

GIST

Gastrointestinal stromal tumor

GSK-3

Glycogen synthase kinase-3

HB-EGF

Heparin-binding epidermal growth factor

HEEBO

Human Exonic Evidence-Based Oligonucleotide

HER2

Human epidermal growth factor receptor 2

HGF

Hepatocyte growth factor

HGSC

High-grade serous carcinoma

HIF-1α

Hypoxia-inducible factor-1 alpha

HIF-1β

Hypoxia-inducible factor-1 beta

HRE

Hypoxia responsive element

HRG

Heregulin

HSP

Heat shock protein

ICM

Intracellular domain

IGF-I

Insulin-like growth factor-I

IOSE

Immortalized ovarian surface epithelium

JM

Juxtamembrane domain

JNK

Jun N-terminal protein kinase

LGSC

Low-grade serous carcinoma

LH

Luteinizing hormone

LOH

Loss of heterozygosity

mAb

Monoclonal antibody

MAPK

Mitogen-activated protein kinase

MDCK

Madin-darby canine kidney

MDM2

Murine double minute 2

MET

Mesenchymal epithelial transition factor

MIP

Molecular inversion probe

MMP

Matrix metalloproteinase

mTOR

Mammalian target of rapamycin

NSCLC

Non-small-cell lung cancer

ODD

Oxygen-dependent degradation

OSE

Ovarian surface epithelium

PAI-1

Plasminogen activator inhibitor-1

PDGF

Platelet-derived growth factor

PI3K

Phosphatidylinositol 3-kinase

PKA

Protein kinase A

PHD

Prolyl hydroxylase

PTEN

Phosphatase and tensin homolog deleted on chromosome 10

PVDF

Polyvinylidene fluoride

RAS

Rat sarcoma

RBD

RAF1-binding domain

RCC

Renal clear cell carcinoma

RD

Regulatory domain

RING

Really interesting new gene

ROS

Reactive oxygen species

RTK

Receptor tyrosine kinase

SD

Standard deviation

SDS

Sodium dodecyl sulphate

xiv

siRNA

Small interference RNA

SIAH

Seven in absentia homolog

SNP

Sodium nitroprusside

SOS

Son of Sevenless

STAT

Signal transducer and activation of transcription

STIC

Serous tubal intraepithelial carcinoma

Sp1

Stimulating protein 1

SPRY

Sprouty

TCGA

The Cancer Genome Atlas

TESK1

Testicular protein kinase 1

TGFα

Transforming growth factor alpha

TGFβ

Transforming growth factor beta

TKD

Tyrosine kinase domain

TMD

Transmembrane domain

TKI

Tyrosine kinase inhibitor

uPA

Urokinase-type plasminogen activator

VEGF

Vascular endothelial growth factor

VHL

Von Hippel-Lindau

WT-1

Wilms tumor 1

WT

Wild-type

ZEB1

Zinc finger E-box-binding homeobox 1

Acknowledgements

First of all, I would like to express my greatest gratitude to my supervisor, Dr. Peter C.K.

Leung for his invaluable support, patient guidance and warm encouragement during my

study. In addition, I would like to express my sincere gratitude to my supervisory

committee members, Drs. Geoffrey Hammond, Blake Gilks, Y.Z. Wang and Mark Carey

for their scientific criticisms and advice and my experiments and thesis. I would also

want to thanks Drs. Blake Gilks, David Huntsman and Alice S.T. Wong from the

University of Hong Kong for their help and opinions of my experiments.

I also would like to thank Dr. Christian Klausen and Ms. Roshni Nair for their help over

the years.

Here I also thank the Interdisciplinary Women’s Reproductive Health Research Training

Program providing me scholarship.

Lastly, I wish to thank my parents and family members for their love, understanding and

endless support throughout my study.

Chapter 1 Introduction

  • 1.1 Ovarian cancer

Ovarian cancer is the second most prevalent gynaecological malignancy (after

endometrial cancer) among women in the United States (1). A woman’s lifetime risk of

developing ovarian cancer is 1 in 70 (2). Ovarian cancer is the most lethal of all

gynaecologic malignancies with a 5-year survival rate of 30% – 40% (3), and overall

survival has not changed in decades (4). The poor patient outcome is mainly due to the

lack of a reliable screening test for early disease detection. Most ovarian carcinomas are

diagnosed at a late stage, after invasion to the intra-peritoneal cavity, and are thus

inoperable (3). In addition, many ovarian carcinomas respond poorly to therapy (1, 5) or

recur with the development of chemoresistance (1).

Based on their origins, approximately 90% of all human ovarian cancers are

categorised as epithelial ovarian carcinomas (EOCs), which originate in the ovarian

surface epithelium (OSE), and the rest are derived from granulosa, stromal or germ cells

(3). However, evidence supporting an origin of these tumors outside the ovary is

accumulating. Therefore, the paradigm of the OSE as the precursor of EOCs is being

challenged, and the cellular origin of EOC remains controversial.

  • 1.2 Tumorigenesis of EOCs

Fathalla proposed the ‘incessant ovulation theory’ in 1971, suggesting that repeat

ovulation, surface rupture and subsequent repair lead to the trapping of the OSE in the

ovarian stroma and formation of inclusion cysts. Inclusion cysts secrete somatic growth

factors that lead to cell proliferation, genetic aberrations and finally malignant

transformation (6). This hypothesis is supported by substantial epidemiological data. One

case–control study of 150 ovarian cancer patients under the age of 50 years demonstrated

that the risk of ovarian cancer decreased with increasing numbers of live births,

increasing numbers of incomplete pregnancies and the use of oral contraceptives (7).

Another prevailing hypothesis addressing the development of ovarian cancer was

proposed by Cramer and Welch in 1983. Their ‘gonadotropin theory’ proposed that

excessive gonadotropin stimulation contributes to ovarian carcinogenesis (8). The risk of

ovarian cancer increases during the perimenopausal period, when serum gonadotropin

levels peak and thereafter remain elevated (9, 10). Moreover, only 10% – 15% of tumors

appear in premenopausal women (11). Likewise, polycystic ovary syndrome patients

(with high luteinising hormone levels) are more prone to ovarian cancer (12).

Epidemiologic evidence supports the idea that pregnancies, breast feeding, and oral

contraceptive use, which suppress pituitary gonadotropin secretion, reduce the risk of

ovarian cancer (13-16). Mesothelial OSE has been proposed to undergo Müllerian

differentiation during ovarian carcinogenesis. As a result, the OSE loses its mesothelial

characteristics and acquires the properties of the Müllerian system (3). This characteristic

readily explains why histological and immunocytochemical analyses of EOCs including

serous (Fallopian tube), endometrioid (endometrium), clear cell (vaginal) and mucinous

(endocervix) reveal characteristics of Müllerian epithelia rather than mesothelial tumors

(3). For instance, serous carcinomas express PAX2 or PAX8, which are normally

expressed in Fallopian tube epithelia (17, 18). However, the similarities of EOCs to

Müllerian tumors support the possibility of a Müllerian lineage and thus provide an

alternative theory of EOC histogenesis (4, 19). For instance, instead of OSE, serous

carcinomas in Fallopian tube are proposed to be precursors of high-grade serous tumors

(20). On the other hand, endometrioid and clear cell carcinomas are suggested to arise

from endometriosis (21).

1.3 Classification of EOCs

Rather than a single entity, EOCs are heterogeneous diseases comprising tumors

of different subtypes. Serous (high-grade and low-grade), endometrioid, clear cell and

mucinous are the major subtypes of EOCs. Their distinct genetic abnormalities and

oncogenic pathways and differential responses to chemotherapy (1, 22) emphasise the

importance of subtype diagnosis and subtype-specific therapies (23).

1.3.1 High-grade serous carcinoma (HGSC)

HGSC is the most common type of EOC and accounts for 60% of EOC cases and

90% of all serous carcinomas (1). HGSCs are usually diagnosed at an advanced stage

(22) 85% of patients present with widespread peritoneal metastases (1). Accurate

diagnosis of HGSC depends on squamous differentiation and areas with solid growth

(22). For problematic cases, Wilms tumor 1 (WT-1) is an useful immunomarkers due to

its specific expression in HGSCbut not other EOCs (24).

Almost all HGSCs harbour TP53 mutations (up to 97.6%) (25) (Fig. 1.1).

Additional mechanisms including TP53 loss of heterozygosity (LOH) (26) and MDM2 or

MDM4 (specific p53 inhibitors) amplification (25) contribute to aberrant p53 expression.

Although p53 dysfunction is a ubiquitous feature of HGSCs, the data regarding the

prognostic value of p53 are conflicting (25, 27).

Other characteristic genetic abnormalities of HGSCs include loss of BRCA1 and

BRCA2 (Fig. 1.1). Both BRCA genes can be inactivated through germline or somatic

mutations, and BRCA1 is also silenced epigenetically through promoter hypermethylation

(28), leading to familial and sporadic HGSC. Among BRCA-related hereditary ovarian

cancers, up to 57% -100% are HGSCs (22, 29), highlighting the importance of BRCA in

HGSC tumorigenesis.

Owing to their role in DNA repair, BRCA and p53 dysfunction lead to genomic

instability and allow accumulation of further genetic alterations along the path of tumor

progression. HGSCs normally display aneuploidy and intratumoral genetic heterogeneity

(30).

In addition to the conventional theory of development of EOCs from the OSE or

cortical inclusion cysts (3), there is an emerging view that EOCs may derive from cells

outside the ovary, which subsequently implant and expand within the ovary and present as

tumors originating in ovary (4). This mechanism may explain why the OSE has a

mesothelial phenotype whereas HGSCs have Müllerian morphology and express a

Müllerian marker (PAX8) but not mesothelial markers (18). Serous tubal intraepithelial

carcinomas (STICs) in the fimbriated end of Fallopian tubes are proposed to be the

precursors of HGSCs of the ovary (20). In addition to the high incidence of dysplastic

changes in the Fallopian tubes of women with a genetic predisposition for familial

ovarian cancer (31), up to 50% - 60% of HGSC patients also have STICs (32, 33). A

clonal relationship between STICs and HGSCs is further supported by identical TP53

mutations in the tumors (34) (Fig. 1.1). A gene profiling study showed that HGSCs and

the Fallopian tube epithelium share similar gene expression profiles, and HGSCs were

found to be less closely related to the OSE (35), suggesting that HGSCs may originate

from the Fallopian tube epithelium rather than the OSE.

Surgery is the primary treatment for all ovarian cancers including HGSC.

Although the majority (70% - 80%) of HGSC patients show initial responses to

platinum/taxane-based chemotherapy (1, 22), 70% of them experience a recurrence, and

many recurrent tumors become resistant to etoposide and doxorubicin (1). All of these

factors together, combined with the late diagnosis, contribute to the poor prognosis of

HGSC patients, who have a 10% - 20% 5-year survival rate (1).

1.3.2 Low-grade serous carcinoma (LGSC)

Among all ovarian serous cancers, 10% are LGSCs. Differentiation diagnosis

between LGSC and HGSC is usually based in the uniformity of nuclei. In addition,

psammoma bodies, differentiated architecture with papillary growth are characteristic

feathers of LGSC (22). The prognosis of LGSC patients is better than that for HGSC

patients (1). Rarely, LGSCs are found concurrently with HGSCs, suggesting the

progression of LGSC to HGSG (36) (Fig. 1.1). However, it is well accepted that low-

grade and high-grade serous carcinomas are fundamentally distinct tumors (22). LGSCs

rarely harbour TP53 or germline BRCA mutations (1, 22). Instead, activating mutations of

KRAS, BRAF, (37) or ERBB2 (38) are common. Accordingly, 60-70% of LGSCs express

active MAPK (39), highlighting the role of the KRAS/BRAF/MEK/MAPK pathway in

LGSC pathogenesis. Mutations of KRAS and BRAF are also found in serous borderline

tumors, which are believed to be the immediate precursors of LGSCs (1) (Fig. 1.1).

1.3.3 Endometrioid carcinoma (EC)

EC is the second most common histologic subtype of EOC, accounting for 10% -

20% of cases (40). ECs are mostly diagnosed at stages I and II, which accounts for the

better prognosis than other subtypes (22). EC are commonly characterized with squamous

or mucinous differentiation (22). Some clinicians diagnosed high-grade carcinomas with

glandular differentiation as EC, however, this diagnostic is doubted as glandular

differentiation is also involved in serous neoplasia (22).

Similar to high-grade serous, high-grade EC may arise de novo or by rare stepwise

progression from borderline and low-grade tumors (40). High-grade de novo ECs

typically have mutations in TP53 (41) (Fig. 1.1). On the other hand, the genetic defects

found in borderline or low-grade endometrioid tumors are also commonly found in high-

grade tumors. For instance, CTNNB1 (38% - 50% of cases) and PTEN (phosphatase and

tensin homolog deleted on chromosome 10) (20%) mutations have been identified in

both, suggesting a precursor role of these lesions (22, 42) (Fig. 1.1). The CTNNB1 gene

mutation encodes a β-catenin protein that is resistant to degradation, which allows

stabilization and nuclear accumulation of β-catenin (43) and can cause endometrioid

carcinogenesis. There is also evidence suggesting that endometriosis gives rise to EC.

ECs share similar molecular signatures with adjacent endometriosis, including LOH of

chromosome 12 (44) and 10q23 (45) mutations in ARID1A tumor suppressor gene, which

encodes adenine-thymine (AT)-rich interactive domain-containing protein 1A (21) and

PTEN (45). Furthermore, up to 42% of EC cases are associated with endometriosis (46),

and 67% - 100% of endometriosis cases are associated with coexisting EC (19) (Fig. 1.1).

1.3.4

Clear cell carcinoma (CCC)

CCCs account for approximately 10% of all EOC cases. Though CCC patients are

usually diagnosed at stages I or II, chemo-resistance, relapse after surgery and the

aggressiveness of CCCs make the prognosis of CCC patients worse than that of other

ovarian carcinomas at early stages (1, 22). The presence of clear cells is not sufficient for

a diagnosis of CCC. Additional criteria for CCC include papillary/tubulocystic

architecture and low mitotic rate (22).

In sharp contrast to HGSCs, most CCCs contain wild-type TP53 (47) and

BRCA1/2 (48). CCCs are best characterised by aberrant activation of PI3K signalling,

including activating mutations of PI3KCA (42, 47), loss of PTEN expression (49) and

activated mTOR (mammalian target of rapamycin) (50) (Fig. 1.1). Recently, two groups

demonstrated frequent somatic mutation of the ARID1A tumor suppressor gene in CCCs

(21, 51). Furthermore, ARID1A mutations are found in CCCs as well as in adjacent

atypical endometriosis, but not in distant endometriosis (21). This difference implicates

ARID1A mutations as early events in the transformation of endometriosis into cancer and

strengthens the association between endometriosis and clear cell carcinogenesis.

  • 1.3.5 Mucinous tumors

Although mucinous tumors constitute 10% - 15% of all primary ovarian tumors,

most of them are benign and borderline tumors. Only 3% - 4% are mucinous carcinomas

(22), making them one of the rarest types of EOCs (4). Most of these tumors show

gastrointestinal differentiation and contain goblet cells, which are key features for their

diagnosis. However, mucinous tumors are often heterogeneous, making adequate

sampling for histological examination critical (22).

The origin of mucinous tumors are unclear, and based on their non-Müllerian

phenotype, development from cortical inclusion cysts is unlikely (4). KRAS mutations are

the most frequent genetic defects in mucinous carcinomas (85% of cases) and represent

an early event in mucinous tumorigenesis. In addition, the ERBB2 gene is amplified in

15% to 20% of mucinous carcinomas (22) (Fig. 1.1).

1.4 Epidermal growth factor (EGF) and the EGF receptor (EGFR)

Numerous steroids, growth factors and cytokines secreted by ovary stromal or

cancer cells promote neoplastic transformation and progression of ovarian cancer (52). Of

these, EGF-like growth factors and their cognate receptors have been the most

extensively studied.

1.4.1 EGFR structure and signaling

The EGFR, also known as ERBB1/HER1, together with HER2/neu,

ERBB3/HER3 and ERBB4/HER4 comprise the ERBB (or HER) family (53). All four

members are Type I transmembrane tyrosine kinase receptors that consist of an

extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain

(ICD) (Fig. 1.2). The ICD can be functionally further divided into i) the juxtamembrane

domain (JD), which mediates EGFR internalisation and signalling specificity (54, 55); ii)

the tyrosine kinase domain (TKD), which possesses intrinsic kinase activity (56); and iii)

the carboxy-terminal regulatory domain (RD). Upon ligand binding, the ECD undergoes a

conformational change that allows dimerization of ERBBs (Fig. 1.2). Dimerization can

be homodimerization or heterodimerization between various ERBBs (57) and HER2 is

the most common binding partner for ERBBs (58). In the dimer complexes, the TKD

undergoes tyrosine phosphorylation. Subsequently, tyrosine residues in the RD are

transphosphorylated, in turn recruiting diverse cytoplasmic adaptor proteins and

activating a variety of signalling pathways (53). In ERBB3, a single amino substitution in

the TKD abrogates the intrinsic kinase activity, making ERBB3 homodimers inactive and

the signal transduction of ERBB3 relies on heterodimerization with other ERBB members

(56).

The ERBB family members invariably activate the ERK cascade through

recruiting the adaptor proteins Grb2 or Shc (59) (Fig. 1.2). The PI3K/AKT pathway is

triggered by most activated ERBB dimers, which have different kinetics and potency.

Such differences are probably due to the fact that ERBB3 and ERBB4 are capable of

direct binding to the PI3K p85 regulatory subunit, whereas the EGFR and HER2 couple

indirectly with p85 through adaptor proteins or ERBB3/ERBB4 (60). Phosphorylated-

AKT was detected in 68% of ovarian carcinomas on a tissue microarray (61). Activation

of these pathways in cancer has been associated with increased cell survival, growth,

angiogenesis and metastasis (57, 62). Furthermore, aberrant levels of the EGFR and

HER2, as well as their downstream ERK and AKT, lead to resistance to platinum-based

chemotherapy (61, 63, 64).

1.4.2 EGFR expression and mutations in ovarian cancer

Aberrant EGFR activity is achieved through a variety of mechanisms, including

overproduction of ligands, overexpression of receptors and constitutive activation of

receptors through mutation or loss of regulators (57). EGFR overexpression and mutation

are frequent events in human malignancies; for example, the EGFR gene is amplified in

up to 40% of gliomas (65). There are many published reports regarding EGFR expression

and its prognostic significance in ovarian cancer. In general, increased EGFR is

correlated with more aggressive diseases and poorer patient outcomes (53). In various

studies, the frequency of immunohistochemical detection of EGFR expression in ovarian

cancer tissues ranges from 4% - 100% (53). EGFR gene amplification is seen in 43% of

serous carcinomas (66), and EGFR overexpression is more common in serous (66, 67)

and endometrioid (68) carcinoma subtypes. Both a higher gene copy number and protein

expression correlate with the serous subtype (66). Higher levels of the EGFR are found in

omental metastases (69) and carcinomas of advanced stages (68, 70), implicating a role

for the EGFR in tumor progression and metastasis. This role may explain the fact that

high EGFR expression correlates with unfavourable outcomes in terms of overall survival

(66-68, 71) and disease-free survival (67). Increased EGFR expression is also associated

with carcinomas (compared with LMP or benign tumors) (70, 72), higher tumor grade

(66, 73), higher proliferative index (66, 73), larger residual tumor size (66, 68), and

advanced age of patients (66, 74). However, data from numerous studies are highly

variable and contradictory; many investigators have found no relationship between the

EGFR and the clinical features and prognostic factors mentioned above (53).

In addition to gene amplification and protein overexpression, EGFR hyperactivity

may arise from EGFR gene mutations (75). An 801-base pair in-frame deletion in the

extracellular domain of the EGFR results in a constitutively active EGFR variant III

(EGFRvIII) mutant. Independent of ligands, this mutant is constitutively active in terms

of receptor dimerization, autophosphorylation and downstream signalling activation (75,

76). EGFRvIII is expressed in a large proportion of gliomas and breast carcinomas and

75% of ovarian carcinomas (77). However, other reports showed that EGFRvIII is rare in

EOC (66, 78) Expression of this mutant in an EOC cell line causes epithelial-

mesenchymal (EMT) transition, cell migration and metastasis (79). Mutations in the

catalytic domain (exon 18, 19, 20 or 21) of EGFR are less frequent in ovarian carcinomas

(66, 74, 80). Activating mutations of the EGFR enhance the sensitivity of lung cancer

patients to EGFR inhibitors (81). In ovarian cancers patients, responses to gefitinib (a

small molecule tyrosine kinase inhibitor) have been reported to be independent of EGFR

mutational status in one study (82), but another reported that EGFR-activating mutations

have also been reported to confer patient responses to gefitinib and lead to longer

progression-free survival (67).

In contrast to numerous investigations of EGFR expression and mutations, only a

few studies regarding EGFR activation status in ovarian carcinomas have been published.

These studies reported values for EGFR activation, as detected by immunohistochemistry,

of 11.8%, 35% and 100% (62, 83, 84). EGFR phosphorylation was also found to be

positively correlated with a metastasis-promoting protein (matrix metalloproteinase 9)

and negatively correlated with E-cadherin, which inhibits metastasis (84).

1.4.3 EGFR ligands

The activation of ERBBs is mediated through ligand-dependent mechanisms.

Ligands of ERBBs contain an EGF-like domain and three intramolecular disulphide

bonds and are synthesised as plasma membrane-bound precursors (85). Based on their

binding specificity, they are classified into four groups: EGF, transforming growth factor-

α (TGF-α) and amphiregulin (AREG) bind the EGFR exclusively; epiregulin (EPI),

betacellulin (BTC) and heparin-binding-EGF (HB-EGF) have dual specificity and bind

the EGFR and ERBB4; and the heregulin (HRG) family constitute the remaining two

groups, which bind both ERBB3 and ERBB4 (HRG-1 and HRG-2) or exclusively to

ERBB4 (HRG-3 and HRG-4) (53). No HER2-specific ligand has been identified yet (57).

Most of these ligands are overexpressed in and correlate with the clinical features

of different types of cancers, including ovarian cancer (57). EGF has been detected in

borderline, low-grade tumors, carcinomas and EOC cell lines (53, 86). Furthermore, up to

72% of epithelial ovarian tumors that are examined are immunopositive for EGF, and a

higher EGF expression level has been found in mucinous cystadenocarcinomas compared

with that in LMP mucinous tumors (87). The EGFR-exclusive ligand TGF-α is frequently

detected in EOCs, with one study showing expression in 77% of tissues examined (53).

The expression of TGF-α correlates with disease grade and stage (86). Concomitant high

TGF-α levels and peak incidence of the disease in post-menopausal women suggest the

importance of this growth factor in ovarian cancer pathology (88). HB-EGF expression is

significantly higher in ovarian cancer than in normal ovaries (89, 90). Furthermore,

elevated HB-EGF protein levels were detected in ascites fluid from ovarian cancer

patients compared with that in the peritoneal fluid of normal women (90). A higher HB-

EGF expression level is significantly associated with shorter progression-free survival of

patients (89). Further, the levels of AREG found in ovarian cancer cell lines, tissues and

the peritoneal fluid of ovarian cancer patients are higher than those of TGF-α and EGF

(89, 91, 92). To date, however, no studies have reported the expression of the other two

EGFR-binding ligands, EPI and BTC, in ovarian carcinomas tissues (53).

It is important to note that the ERBB family members can also be transactivated

by heterologous signals including those from G protein coupled receptors, independent of

their cognate ligands (93-96). Cross activation of ERBBs can be achieved through direct

phosphorylation or induction of ectodomain shedding of ligands (90). For example, the

chemokine CXCL1 transactivates the EGFR through proteolytic cleavage of HB-EGF,

leading to MAPK activation and the proliferation of ovarian cancer cells (97).

1.4.4 Functions of the EGFR and it ligands in ovarian cancer

EGF and TGF-α are the most well-studied ligands of the EGFR. They form

autocrine loops with the EGFR and have numerous regulatory functions in OSE and

ovarian cancer cells. Gene profiling studies of rat OSE have demonstrated that genes

involved in the cell cycle, proliferation, and apoptosis are targets of EGF (98). EGF

treatment increases the proliferation of human primary OSE cells and ovarian cancer cells

in culture (99, 100). In vitro treatment with TGF-α increased cell proliferation in eight

ovarian cancer cell lines in one study (101). TGF-α is also synthesised by cancer cell

lines (102), and disruption of the TGF-α/EGFR autocrine loop by a TGF-α neutralising

antibody or antisense oligodeoxynucleotides blocks proliferation (102, 103). Accordingly,

the in vivo growth of inoculated tumors is also blocked by TGF-α antibody treatment

(104). Interestingly, growth regulation by AREG is unique, as AREG has a biphasic effect

on OSE and ovarian cancer cells (105).! Ovarian cancer cell lines stably transfected with

an antisense EGFR also show decreased proliferation (106). With its potent mitogenic

potential, the EGFR and ligand autocrine loops confer a selective advantage for cells and

are implicated in clonal expansion and in the accumulation of mutations within tumors.

In addition to cell growth, the EGFR and its ligands are implicated in metastasis.

In response to EGF treatment, OSE cells (107) and ovarian cancer cells undergo EMT

(108, 109) and EMT-associated N-cadherin and vimentin expression changes (108). It is

important to note that during EMT, cells adopt a fibroblastic phenotype with reduced

intercellular contact and increased cell motility. Consistent with these observations, tumor

cell migration and invasion have been repeatedly demonstrated to be stimulated by EGF

(84, 99, 108, 110, 111). Motility may be modulated through regulating the integrin

system, which comprises transmembrane receptors forming bridges between the

extracellular matrix (ECM) and the cytoskeleton and plays roles in adhesion and

migration. EGF activates STAT3 signalling and regulates the levels of integrin α2, α6

and β1 (108). Alternatively, invasiveness may be acquired through promoting proteolytic

degradation of the ECM; indeed, EGF increases the expression of uPAR (urokinase-type

plasminogen activator receptor) (110) and MMP9 (84, 111). Furthermore, MMP9 is

responsible for the EGF-induced loss of E-cadherin and disruption of adhesion junctions,

leading to a migratory and invasive phenotype (84). In contrast, silencing of the EGFR

suppresses integrin expression, MMP9 activity, cell adhesion and the invasion of ovarian

cancer cells (106, 112). However, no studies on the role of AREG, EPI or BTC in ovarian

cancer cell invasion have been published.

Furthermore, blocking the EGFR suppresses in vivo ovarian cancer cell

tumorigenicity in nude mice (106, 113). EGF also plays a role in regulating the sensitivity

of ovarian cancer cells to cisplatin in vitro (114), and dominant-negative EGFR can

partially restore cisplatin sensitivity in drug-resistant ovarian cancer cells (113).

1.4.5 Clinical activities of EGFR-targeted therapies.

Agents disrupting EGFR activities have been evaluated for their feasibility as

ovarian cancer therapies, for examples, small molecule tyrosine kinase inhibitors (TKIs)

targeting EGFR kinase domain and monoclonal antibodies (mAb) binding EGFR ECD.

Generally, these therapeutics have very limited clinical activities in ovarian cancers (59).

Gefitinib and erlotinib are EGFR TKIs which inhibit ovarian cancer cells and xenografts

growth. No patients showed complete response or partial response in a phase II clinical

trial of gefitinib (83) and only one objective response (4 % of cases) reported in another

trial (74). In a phase II trial of erlotinib, 6% of patients demonstrated partial response and

no complete response has been observed (59). Furthermore, trials have been performed

using TKIs in combinations with cytotoxic chemotherapies like docetaxel and carboplatin

(115, 116). However, none of these treatment regimes showed improvement in clinical

activities (115, 116). On the contrary, significant clinical response to gefitinib and

erlotinib have been observed in 10 % - 30 % NSCLC patients (117). In addition to TKIs,

results from clinical trials of EGFR mAbs are also disappointing. These antibodies block

ligand binding and receptor dimerization, they also reduce EGFR levels through

promoting EGFR internalization and degradation (116). The first clinically tested EGFR

mAb, cetuximab, showed partial response in 4 % of patients (118). In the phase II trial of

another EGFR antibody, matuzumab, neither partial response nor complete response has

been demonstrated (119).

1.5 Sprouty (SPRY)

In the past decade, a novel family of cytoplasmic proteins called SPRY have been

shown to play potent regulatory roles during Drosophila and mouse development (120-

122). Among these studies, the overexpression of SPRY has been shown to mimic the

effects of EGFR signalling loss and prevent EGFR-induced phenotypic changes (121).

Many additional studies have demonstrated that SPRY regulates the RAS/RAF/ERK

pathway that is activated by various RTKs (122-125).

1.5.1 SPRY structure

One Drosophila and four mammalian SPRY homologs have been identified with

conserved structural characteristics, namely SPRY1, SPRY2, SPRY3 and SPRY4 (121,

122, 126). All SPRY members share a conserved C-terminal cysteine-rich region, with

44% – 52% identity between Drosophila spry and mouse Spry1, 2 and 4 proteins (122).

They also contain a conserved SPRY domain that mediates binding to RAF1 (RAF1-

binding domain, RBD) (127) (Fig. 1.3). The C-terminal domain is responsible for plasma

membrane localisation (121, 128, 129). Deletion of the C-terminal domain abolishes

membrane localisation and many of the biological functions of SPRY (121, 125, 128). In

contrast, the N-terminal domains of the SPRY proteins are more divergent and are

proposed to account for the substrate diversity and specificity of the individual SPRY

members (122). However, the N-terminal domains of all SPRY proteins invariably

contain a conserved tyrosine residue (Fig. 1.3). The tyrosine is phosphorylated upon

growth factor stimulation and mediates membrane translocation. Most of the functions of

SPRY1 and SPRY2 (126, 130, 131) but not SPRY4 (126) are dependent on the

phosphorylation of this tyrosine.

1.5.2 SPRY functions and mechanisms

It is well known that downstream of various RTKs, SPRY proteins specifically

inhibit the RAS/RAF/ERK pathway without affecting the p38, JNK or PI3K/AKT

pathways (124, 125). However, there is still no consensus on how SPRY blocks ERK

activation. Drosophila spry regulates ERK signalling at a point downstream of EGFR and

upstream of RAS and RAF (121). In mammalian cells, by direct interaction with Grb2,

SPRY1 and SPRY2 decrease RAS activation, RAS-RAF binding and RAF activation

(124) (Fig. 1.3). Alternatively, downstream of RAS and independent of RAS activation,

SPRY2 inhibits FGF-induced RAF activation (125). Interestingly, SPRY is only capable

of binding to wild-type RAF, but oncogenic RAF is refractory to SPRY inhibition,

leading to unchecked ERK activation in cell harbouring oncogenic RAF mutations (132).

The evidence to date suggests the existence of multiple mechanisms that depend on the

cellular context and/or the identity of the RTK (127).

In contrast to initial observations (124, 125), accumulating evidence supports the

mechanism that the PI3K/AKT cascade is also a target of SPRY (133, 134). To date,

there has only been one study on the mechanism of AKT repression by SPRY, which

demonstrated that SPRY2 increases the amount of PTEN and decreases its

phosphorylation, leading to a decreased activation of AKT by EGF.

In contrast to SPRY2, the understanding of the regulatory mechanisms of SPRY 4

is extremely limited and conflicting. SPRY4 was first reported to represses RTK-induced

ERK phosphorylation upstream of, or parallel to, RAS (135, 136). However, later studies

found that mouse SPRY4 interferes with the VEGF-induced RAS-independent activation

of RAF1 through a direct association with RAF1 (137). SPRY4 can also interact with

TESK1 (testicular protein kinase 1) to inhibit growth factor-induced RAS/MAPK

signalling (136, 138) (Fig. 1.3).

Although SPRY proteins are generally considered to be inhibitory, SPRY2 may

enhance EGF signalling in a cell type-specific manner (127). Cbl proteins are E3

ubiquitin ligases that recognise, ubiquitinate and target RTKs for degradation (139-141).

Through direct interaction with the Cbl RING finger domain (142, 143), SPRY2 is

capable of sequestering Cbl and thereby protects EGFR from degradation (144), resulting

in higher EGFR levels and sustained activation (127) (Fig. 1.3).

1.5.3 Regulation of SPRY activity

The feedback nature of SPRY arises from the prompt activation of SPRY by RTK

activity. In addition to phosphorylation and membrane translocation as described above,

the transcriptional regulation of SPRY is also important. During Drosophila embryonic

development, SPRY level is higher in cells that are responsive to FGF and EGF (121,

123, 145, 146). Levels of SPRY2 and SPRY4 have been shown to be upregulated by

activation of RAS/ERK signalling upon EGF and FGF stimulation in vitro (147).

Similarly, in cancer cells, SPRY level is induced by growth factors (148) and oncogenic

mutations downstream of RTK (149).

1.5.4

SPRY expressions in cancer

Aside from a few exceptions (132, 150), the expression of SPRY has been found

to be downregulated in cancer cells in most reports. SPRY2 and/or SPRY1 are

significantly down-regulated in breast (151), prostate (152, 153), endometrial (154), and

liver (155) cancers. The effects of the altered expression of different SPRY isoforms is

likely cancer type-specific. The same group recently demonstrated a lower level of

SPRY2, but not SPRY1, in hepatocellular carcinoma (155). Furthermore, the mechanisms

of downregulation are also variable among different types of cancers. For example, in

prostate cancer, SPRY2 is silenced through both epigenetic promoter hypermethylation

and LOH (156). In contrast, SPRY2 loss in breast and liver cancer is independent of

promoter methylation (151, 155). The prognostic value of SPRY has been demonstrated

in renal (157), liver (158) and prostate (153) cancer patients. Moreover, SPRY4 mRNA

level is as a reliable marker of the response of gastrointestinal stromal tumors to Gleevec

treatment (150).

  • 1.5.5 SPRY as tumor suppressor genes

The tumor suppressor role of SPRY is supported by its negative effects on

tumorigenesis. SPRY have been reported to inhibit proliferation (151, 155, 159, 160),

migration and invasion (148, 159-161), cell cycle progression (148), in vitro and in vivo

tumorigenesis (148, 151) and metastasis (162).

1.6 Hypoxia-inducible factor-1 alpha (HIF-1α)

Hypoxia, a low oxygen tension condition, is a common phenomenon in solid

tumors and is a driving force of tumor progression. Although severe or prolonged hypoxia

is toxic to cancer cells, it selects cancer cells that with adaptive and genetic changes that

allow them to survive and proliferate (163). Most biological changes in response to

hypoxia are mediated through the induction of the critical molecular mediators of

hypoxia, the hypoxia-inducible factors (HIFs). Following stabilization and nuclear

translocation in hypoxic conditions, HIF binds to hypoxia-response elements (HREs) to

induce expression of numerous hypoxia-response genes (Fig. 1.4), thereby regulating

multiple steps of tumorigenesis, including angiogenesis, metastasis and resistance to

therapy (163). These processes contribute to the malignant phenotype, increase the rate of

mutation and decrease overall patient survival (164).

1.6.1 HIF-1α structure

The HIF family, including HIF-1, HIF-2 and HIF-3, belongs to the PAS (PER-

ARNT (arylhydrocarbon receptor nuclear translocator)-SIM) family of basic helix-loop-

helix (bHLH) transcription factors. HIFs bind to DNA as heterodimers of a constitutively

expressed HIF-1β subunit, also known as ARNT, and an oxygen-dependent α subunit

(HIF-1α, HIF-2α and HIF-3α). Structural analyses revealed that HIF-1α contains a bHLH

domain (for dimerization and DNA binding), a PAS domain (for dimerization and target

gene specificity), two transactivation domains (N-terminal, NTAD and C-terminal,

CTAD) and an oxygen-dependent degradation (ODD) domain (Fig. 1.4). The ODD

domain is required for the ubiquitin–proteasome degradation pathway under aerobic

conditions, and due to the absence of the ODD domain, HIF-1β is constitutively

expressed in normoxia.

1.6.2 HIF-1α regulation

The abilities of HIFs to act as the master regulator of cellular responses to

hypoxia and in O 2 -homeostasis arises from the tight regulation of its activities by O 2

availability. The activities of HIFs are largely controlled through the availability of the α

subunit, whose half-life is extremely short under normal oxygen tension (165). Therefore,

the majority of investigations focus on the alpha subunits, and HIF-1α is the most

extensively studied. In addition to tissue hypoxia, HIF-1α stabilization can be regulated

by hormonal stimulation and genetic alterations under normoxic conditions.

1.6.2.1 Hypoxia

Under well-oxygenated conditions, conserved proline residues (Pro402 and

Pro564) within the ODD domain of HIF-1α are hydroxylated by prolyl hydroxylases

(PHDs) (Fig. 1.4). Hydroxylated HIF-1α is recognised and ubiquitinated by Von Hippel-

Lindau (VHL), the substrate recognition component of an E3 ligase complex.

Ubiquitinated-HIF-1α is then targeted to the proteasomal degradation pathway (166). As

the enzymatic activities of PHDs require O 2 as a cosubstrate, under hypoxia, PHDs are

inactive, hydroxylation of HIF-1α is suppressed, and HIF-1α is stabilized (Fig. 1.4).

PHDs are transcriptionally regulated by hypoxia in both HIF-dependent and HIF-

independent pathways, which creates a negative feedback loop (165). Hypoxic induction

of PHD2 and PHD3 is found in a wide range of cell types; however, induction is not

observed in cells lacking HIF-1α or HIF-1β (167). Silencing HIF-1α reduces the

induction of PHD2 and PHD3 by hypoxia (168). The HIF-independent pathway is

mediated by SIAH1 and SIAH2, specific E3 ligases of PHD1 and PHD3 (169, 170),

whose expression is transcriptionally upregulated by hypoxia in a HIF-independent

manner (171). Hypoxia-independent regulation of PHDs by cellular and hormonal factors

such as oncogenic RAS and Src (see below), reactive oxygen species (ROS) (172), TGF-

β (173) and endothelin (174) serves as one of the mechanisms of HIF-1α regulation in

normoxia.

Stabilized HIF-1α translocates into the nucleus and then dimerizes with HIF-

1β to constitute HIF-1. HIF-1 heterodimers activate transcription by recruiting the

transcriptional coactivators p300 and CREB binding protein (CBP). Interaction between

HIF-1α and p300/CBP is regulated by factor inhibiting HIF-1 (FIH-1) in an oxygen-

dependent manner. FIH, which belongs to the 2-oxoglutarate and Fe(II)-dependent

dioxygenase superfamily, hydroxylates the conserved asparagine (Asn803) residue within

the HIF-1α CTAD and prevents HIF-1α/p300 interaction (175). When hypoxia prevents

asparagine hydroxylation by FIH, the CTAD is activated, interacts with p300/CBP and

binds to HREs to regulate hypoxia-regulated gene expression. Therefore, in addition to

HIF-1α stabilization, the complete activation of HIF transcriptional activity also requires

CTAD activation.! Kung et al. showed that blocking the interaction of HIF-1α with

p300/CBP attenuates hypoxia-inducible gene expression, reduces capillary formation and

inhibits tumor growth in nude mouse xenografts (176).

1.6.2.2 Regulation of HIF-1α in normoxia

1.6.2.2.1 VHL mutations

HIF-1α can also be activated in tumors under normoxic conditions through

genetic alterations in the oxygen-signalling pathway. As mentioned earlier, the VHL

tumor suppressor gene product, pVHL, functions as the substrate recognition subunit of

the ubiquitin E3 ligase complex that targets HIF-1α for degradation. This interaction is

dependent on the hydroxylation of one or both of the two conserved prolines (Pro402 and

Pro564) of HIF-1α by PHDs. Due to the O 2 -sensing function of PHDs, VHL loss-of-

function results in HIF stabilization regardless of oxygen tension (177, 178).

Inactivating germline VHL mutations predispose individuals to VHL diseases

including renal clear cell carcinomas (RCCs) and haemangioblastomas (179, 180). Using

immunohistochemistry, HIF-1α and HIF-2α proteins are found to be overexpressed in all

tested RCCs and haemangioblastomas (181). Similarly, cells lacking the VHL wild-type

protein constitutively express HIF-1α protein in normoxia (181, 182), which can be

rescued by the reintroduction of a functional VHL (181). Accordingly, the mRNA of the

hypoxia-regulated genes VEGF (181, 183) and GLUT1 (181, 184) are found to be higher

in VHL-defective cells. Finally, expression of wild-type VHL in VHL-defective RCC cell

lines prevents tumor formation in nude mice (181, 185).

1.6.2.2.2 AKT and PI3K

Ample evidence suggests that PI3K/AKT signalling can also stabilize HIF-1α and

induce HIF-1 activity (186-192). In hypoxic prostate cancer cells, inhibition of PI3K

using pharmacologic inhibitors or siRNA knockdown decreases HIF-1α levels (188, 189)

and VEGF promoter activity (190). The expression of HIF-1α and VEGF and the

angiogenesis of prostate tumor xenografts are inhibited by dominant negative AKT

mutants (189). HIF-1α stabilization by AKT does not require functional VHL (193).

Activation of the PI3K/AKT pathway can arise from amplification of the

phosphatidylinositol 3-kinase catalytic subunit α (PIK3CA) oncogene in human

malignancies, including ovarian cancer (194). PIK3CA level is positively correlated with

VEGF expression in ovarian cancer cell lines. Treatment with a PI3K inhibitor abrogates

HIF-1α and VEGF expression (195). PIK3CA level has also been detected in most

advanced-stage ovarian cancer biopsies and positively correlates with the VEGF level

and the extent of microvascular development (195).

Moreover, components of the PI3K/AKT signalling pathway, both upstream and

downstream, have been shown to be involved in HIF-1α regulation, including PTEN, a

molecular inhibitor of AKT. Glioblastoma cell lines lacking functional PTEN express

high levels of VEGF even in normoxia, which is suppressed by restoration of wild-type

PTEN (187). Overexpression of PTEN in prostate tumor cells reduces HIF-1α levels

(190), VEGF expression, angiogenesis and tumor growth (189). Downstream of AKT,

inhibition of mTOR (mammalian target of rapamycin) reverses the effect of AKT on HIF-

1α and target gene expressions (191, 196).

AKT has been shown to stabilize HIF-1α without impairing prolyl hydroxylation

and HIF-1α degradation, as revealed by antibodies specific for hydroxylated proline

(197). However, alternative mechanisms for normoxic HIF-1α stabilization exist. For

example, AKT positively regulates HIF-1α levels in glioblastoma cells through increased

protein translation (190).

In addition to PI3K and AKT, oncogenes such as RAS (198, 199) and Src (200)

have been implicated in the normoxic activation of HIF-1α. Introduction of the

oncogenic mutant RASV12 and v-Src into cells increases the HIF-1α level and HIF-1

activity. Remarkably, RASV12 and v-Src abolish the hydroxylation of Pro564 in the

ODD domain and hence inhibit HIF-1α degradation. In sharp contrast, cells transfected

with constitutively active AKT upregulate HIF-1α without affecting hydroxylation. This

finding indicates that these oncogenes may stabilize HIF-1α through hydroxylation-

dependent or hydroxylation-independent pathways (197).

1.6.2.2.3 Glycogen synthase kinase 3β (GSK3β )

The AKT target glycogen synthase kinase 3 (GSK3, exists in GSK3α or

GSK3β isoforms) is a negative HIF-1α regulator. Inhibition of GSK3β using LiCl or

GSK3β knockdown increases HIF-1α accumulation and the level of the HIF-1 target PAI-

1 in normoxia, whereas GSK3β overexpression reduces HIF-1α (201). Notably, GSK3β

mediates the balance between HIF-1α stabilization/degradation as a function of the

duration of hypoxia. Mottet et al. showed that short (5 hrs) incubations of prostate tumor

cells in hypoxia lead to AKT activation and the inhibition of phosphorylation of GSK3β

on Ser9, therefore reducing GSK3β activity and resulting in HIF-1α accumulation as well

as increased HIF-1 transcriptional activity. In contrast, prolonged (16 hr) hypoxia

inactivates AKT and hence activates GSK3β, resulting in decreased HIF-1α protein levels

(192). HIF-1α destabilization by GSK3β is via the NTAD is independent of HIF-1α

hydroxylation and pVHL activity (201).

1.6.2.2.4 Heat shock protein 90 (HSP90)

HSP90 interacts with HIF-1α (193, 202, 203) as well as HIF-2α and HIF-3α

(204). These associations are mediated through the HIF-1α PAS domain (203, 205) and

occur in both normoxia and hypoxia (193, 203). Although HSP90 is associated with HIF-

1α in the cytoplasm, HSP90 does not cotranslocate into the nucleus with HIF-1α (206).

The mechanism underlying HIF-1α protection by HSP90 is not yet fully understood and

is thought to occur via VHL-independent degradation (193, 204).

In hypoxia, HSP90 is induced by (206), binds to and protects HIF-1α. Inhibition

of HSP90 by geldanamycin results in loss of HIF-1α accumulation and HIF-1 activation

in hypoxic VHL-defective cells (202, 206).

The level of HSP90 is enhanced by AKT in normoxia. Inhibitors of AKT, but not

MAPK inhibitors, reduce HSP90 expression (193). In VHL-defective RCC cells,

inhibition of the AKT pathway promotes HIF-1α degradation as efficiently as the HSP90

inhibitor geldanamycin (193). As the PI3K/AKT pathway has no effect on HIF-1α

hydroxylation, protection of HIF-1α by HSP90 increases HIF-1α stabilization by AKT,

independent of proline hydroxylation, subsequent VHL ubiquitination and proteasomal

degradation.

In addition to AKT, HSP90 mediates the HIF-1α regulation of the kinase RACK1

(receptor of activated protein kinase C) (207). Moreover, carbon monoxide, an

environmental factor implicated in angiogenesis (208), stabilizes HIF-1α and promotes

VEGF expression through enhancing HIF-1α/HSP90 interaction (209).

1.6.2.2.5 Hormonal regulation

Another common mechanism for the induction of HIF-1α in normoxia involves a

large variety of growth factors and cytokines. EGF (188) and lysophosphatidic acid (210)

induce HIF-1α through AKT activation to stimulate VEGF expression. IGF-II regulates

cell survival through AKT and HIF-1α induction (211). TGF-β regulates the expression

of HIF-1α and the HIF-1 targets PHD2 and PAI (173). Other hormones including IGF-I

(187, 191, 212, 213), PGDF (186) and follicle-stimulating hormone (FSH) (214) have

been reported to regulate the expression of HIF-1α.

1.6.3 HIF-1α expressions in cancer

The expression of HIF-1α has been comprehensively investigated in panels of

human normal and cancerous biopsies by immunohistochemistry (215, 216). Most normal

tissues show no HIF-1α immunoreactivity. In contrast, overexpression of HIF-1α was

detected in 50% – 60% of malignant samples in one study, which represented most types

of tumors examined, including breast, liver, lung, colon, prostate, ovarian, renal and

pancreatic cancer. Furthermore, HIF-1α overexpression was found to be associated with

metastasis and high-grade tumors. Two-thirds of the regional lymph node and bone

metastases were HIF-1α positive. In addition, a high level of HIF-1α was more

commonly found in breast metastases than in primary tumors. Among brain tumors of

different grades, the strongest HIF-1α expression level was detected in the most

malignant and vascularised tumors (215). Both borderline ovarian tumors and epithelial

ovarian cancers were positive for HIF-1α expression (217, 218). HIF-1α has been shown

to be a negative prognostic factor in most cancers (215, 219, 220)

1.6.4 HIF-1α functions in cancer

1.6.4.1 Angiogenesis

When solid tumors expand, tumor hypoxia increases due to high metabolism and

excessive oxygen consumption as well as the increased distance between tumor cells and

local capillaries (221). To promote blood vessel formation and sustain growth in hypoxic

microenvironments, tumors transition from a nonangiogenic to angiogenic phenotype,

termed the “angiogenic switch” (222). As a prime physiological regulator of the

angiogenic switch (223), a positive correlation has been found between HIF-1α

overexpression and tumor vascular density (215) in brain and ovarian (217) tumor

biopsies.

During the angiogenic switch, hypoxia shifts the balance toward proangiogenic

factors and reduces the expression of antiangiogenic factors such as thrombospondin-1

and thrombospondin-2 (224). To actively promote angiogenesis, HIF activates the

expression of various angiogenic proteins including growth factors, cytokines, and a

number of small molecules, including the key angiogenic factor VEGF. VEGFR1 and

VEGFR2 on endothelial cells mediate the mitogenic effects of VEGF on endothelial cells.

Therefore, VEGF has strong angiogenic activity, and treatment with a VEGFR inhibitor

results in a 60% reduction in tumor vasculature in mouse models (225).

The VEGF promoter contains HRE binding sites and is a direct transcriptional

target of HIF-1 (226). Accordingly, xenografts of HIF-1β-deficient hepatoma cells exhibit

reduced levels of hypoxia-induced VEGF mRNA and reduced tumor vascularisation and

proliferation (227). HIF-1α is also expressed in endothelial cells and mediates autocrine

signalling of VEGF/VEGFR2 in endothelial cell proliferation and blood vessel formation

(228). More importantly, human tumors with high HIF-1α and VEGF expression levels

are correlated with more aggressive and malignant phenotypes (229).

In addition to the hypoxia-mediated activation of HIF, the AKT/HIF-1α axis also

mediates VEGF level and angiogenesis stimulated by growth factors (188, 191) and other

hormones (210, 214) under normoxia.

1.6.4.2 Metastasis

Metastasis is the primary cause of cancer-related deaths. It is a multistep process:

from the initial epithelial-mesenchymal transition (EMT), dissemination, and homing to

the final organotropic colonisation. The role of hypoxia and HIF in tumor metastasis is

supported by the observation that HIF-1α expression is upregulated or more commonly

found in metastases and high-grade tumors (215). Mechanistically, hypoxia and HIF are

potent regulators of many key factors that determine tumor cell invasiveness and

metastatic potential, namely E-cadherin, lysyl oxidase (LOX), chemokine receptor

CXCR4 (CXCR4), stromal-derived factor 1 (SDF-1) and plasminogen activator inhibitor-

1 (PAI-1).

HIF-1 promotes metastasis through repressing E-cadherin, a major cell adhesion

molecule that maintains epithelial integrity. Loss of E-cadherin is a hallmark of and

prerequisite for metastasis. Reduced E-cadherin expression is associated with increased

metastasis in patients with ovarian (230), breast (231), prostate (232) and lung cancers

(233). The antimetastatic role of E-cadherin is supported by the finding that restoration of

E-cadherin in cancer cells inhibits metastasis (234). Repressed E-cadherin levels have

been found to be correlated with HIF-1α expression in ovarian (235) and RCC carcinoma

biopsies (236). In hypoxic ovarian cancer cells and VHL-defective RCC cells, HIF-1α is

overexpressed and EMT and cell invasion are increased in vitro (235, 236). E-cadherin in

these cells is repressed by HIF-1α through inducing the E-cadherin-specific repressors

Snail or ZEB1 (235, 236).

HIF also promotes metastasis through modulating the extracellular matrix. LOX is

an enzyme that is involved in extracellular matrix formation. HIF-1 is a potent inducer of

LOX, and secreted LOX contributes to the invasive properties of hypoxic human cancer

cells. The importance of LOX in hypoxia-induced metastasis is directly underscored by

the finding that inhibition of LOX is sufficient to attenuate hypoxia-induced metastasis in

mice. In patients with breast and head and neck tumors, a high LOX expression level is

correlated with hypoxia and poor distant metastasis-free and overall survival (237).

CXCR4 is the most common chemokine receptor that is expressed in tumor cells.

It is a direct HIF target in hypoxic lung, ovarian, breast and renal cell carcinoma cells

(238). Its ligand, SDF1, is highly expressed in common sites of metastasis, including the

lung, bone marrow, and liver (220). Interactions between CXCR4 and its ligand SDF-1

mediate the metastatic homing of tumor cells in response to hypoxia (220, 238)

Another invasion/metastasis related gene PAI-1 is a direct target of HIF-1α. PAI-

1 levels are enhanced through PI3K/AKT and ERK1/2 via HIF-1α in prostate and gastric

cancer cells (212, 239).

1.7 Hypothesis and objectives

Loss of expression or activity of SPRY has been demonstrated in various human

malignancies. Nevertheless, no studies on SPRY in ovarian cancer have been reported.

Given the implications of various RTKs in ovarian tumorigenesis, I tested the hypothesis

that SPRY acts as a tumor suppressor in ovarian cancer. The main objectives of this thesis

are to investigate the expression and functions of SPRY in ovarian cancer, specifically in

the context of EGFR and HIF-1α signalling.

Objective 1. In Chapter 2, I investigated whether the SPRY mRNA is deregulated and

examined the function of SPRY2 in ovarian cancer.

1) The mRNA levels of SPRY members in ovarian cancer tissues and cell lines was

investigated.

2) The incidence of SPRY2 gene deletion was evaluated.

3) The role of SPRY2 in EGF-induced cell invasion was tested.

Objective 2. In Chapter 3, I tested the roles of an alternative EGFR ligand (AREG) and

regulator (SPRY4) in ovarian cancer invasion.

1) The effect of AREG on invasion was tested.

2) The underlying molecular pathways were elucidated.

3) The effect of AREG on SPRY4 level was examined.

4) The effect of SPRY4 on AREG-induced invasion was investigated.

Objective 3. In Chapter 4, I explored the regulation of SPRY4 by EGF and the effects of

SPRY4 on EGF signalling.

1) The role of AKT/HIF-1α in EGF-induced SPRY4 level was tested.

2) The feedback of SPRY4 on the EGF-induced level and activity of HIF-1α was

evaluated.

3) The role of AKT in SPRY4-mediated HIF-1α regulation was investigated.

Objective 4. In Chapter 4, I extended the investigation to the underlying mechanisms of

SPRY4 inhibition of HIF-1α level.

1) The inhibition of HIF-1α level by SPRY4 was confirmed.

2) The level of regulation of HIF-1α by SPRY4 was determined.

3) The roles of PHD in the SPRY4 regulation of HIF-1α level was investigated.

Figure 1.1 The chart illustrates the pathogenesis of epithelial ovarian cancers. Low-grade serous carcinomas arise from

Figure 1.1 The chart illustrates the pathogenesis of epithelial ovarian cancers. Low-grade

serous carcinomas arise from serous borderline tumors containing activating KRAS,

BRAF or ERBB2 mutations. Rarely, low-grade serous tumors progress to high-grade

tumors. High-grade serous carcinomas develop from the Fallopian tube or ovarian surface

epithelium through TP53 and BRCA mutations. KRAS mutations and ERBB2 gene

amplification are frequently found in mucinous tumors. Clear cell carcinomas contain

PI3KCA mutations or loss of PTEN. PTEN and CTNNB1 mutations may lead to the

formation of endometrioid carcinomas. Alternatively, endometrioid carcinomas may

contain TP53 mutations. Endometrioid carcinomas and clear cell carcinomas may also

arise from endometriosis with ARID1A mutations. Modified from Lalwani, N et al, 2011

(1).

Figure 1.2 The structure and signaling of EGFR. EGFR consists of an extracellular domain (ECD), a

Figure 1.2 The structure and signaling of EGFR. EGFR consists of an extracellular

domain (ECD), a transmembrane domain (TMD) and an intracellular domain (ICD). The

ICD can be further divided into a juxtamembrane domain (JD), a tyrosine kinase domain

(TKD) and a regulatory domain (RD). Upon the binding of EGFR ligands, such as EGF,

transforming growth factor α (TGFα) or amphiregulin (AREG), the TKD is

phosphorylated and thereby activated; the RD is then transactivated. The phosphorylated

RD recruits adaptor proteins, including Shc, Grb2 and SOS, and triggers the

RAS/RAF/MEK/ERK cascade. Activated EGFR also stimulates other MAPK pathways

(p38 and JNK) and the PI3K/AKT pathway.

Figure 1.3 A schematic diagram of human SPRY depicting its structure, its interacting partners and the

Figure 1.3 A schematic diagram of human SPRY depicting its structure, its interacting

partners and the corresponding functional consequences. The C-termini of SPRY proteins

are more conserved and contain a RAF1-binding domain (RBD), which mediates the

interaction with RAF and leads to the inhibition of ERK activation. ERK inhibition is

also achieved through SPRY interaction with Testicular protein kinase 1 (TESK1). SPRY

also interfere with the RAS/RAF/MEK/ERK pathway through interaction with Grb2. The

N-termini of SPRY are more divergent but invariably contain a conserved tyrosine

residue. Tyrosine-phosphorylated SPRY binds with Cbl, which inhibits EGFR

degradation and increases EGFR signaling. Interaction between SPRY and Seven in

absentia homolog 2 (SIAH2) promotes the ubiquitination of SPRY and target SPRY for

proteasomal degradation. Modified from Edwin, F et al, 2009 (240).

Figure 1.4 The diagram illustrates the regulation of HIF-1 α . HIF-1 α is mainly regulated

Figure 1.4 The diagram illustrates the regulation of HIF-1α. HIF-1α is mainly regulated

post-transcriptionally. Under normoxia, PHD proteins hydroxylate the two proline

residues within the HIF-1α oxygen-dependent domain (ODD) domain. Hydroxylated

HIF-1α is prone to Von Hippel-Lindau (VHL)-mediated degradation. When PHDs are

degraded by Seven in absentia homolog 2 (SIAH2) or inactivated by hypoxia, growth

factors (GFs), reactive oxygen species (ROS) or oncogenic RAS, HIF-1α degradation is

prevented. In contrast, the PI3K/AKT/glycogen synthase kinase-3β (GSK3β) pathway

regulates HIF-1α independently of PHD activity. When HIF-1α is stabilized, it would

translocate into the nucleus and dimerize with HIF-1β. With the recruitment of the p300

co-factor, the complex recognizes hypoxia-responsive elements (HRE) in the promoters

of target genes, which are implicated in processes such as angiogenesis, invasion,

proliferation and apoptosis.

Chapter 2 Genetic inactivation of Sprouty2 promotes epidermal growth factor-induced E-

cadherin downregulation and invasion in ovarian cancer cells

2.1 Introduction

Ovarian cancer is the fifth leading cause of all female cancer-related deaths and the

most lethal gynaecologic malignancy in North America. Approximately 60% of women

who develop ovarian cancer will die from the disease (2, 241). Although epithelial

ovarian cancer (EOC) includes a majority of all ovarian carcinomas, its origin and

aetiology have not yet been completely elucidated. Current evidence suggests that

malfunction of receptor tyrosine kinases (RTKs), including epidermal growth factor

receptor (EGFR), contributes to the development of EOC (52). EGFR and its ligands play

a critical role in cell proliferation, survival, and tumor metastasis (53, 242). Moreover,

increased expression levels of EGFR-specific ligands have been identified in ovarian

cancer cells (89) and ascitic fluid (90), and EGFR expression levels were found to be

elevated in advanced stage disease and metastases (53).

In addition to RTK abnormalities, the loss of endogenous regulators represents an

alternative mechanism that leads to aberrant RTK activity. During the past decade, a

novel family of cytoplasmic proteins, Sprouty (SPRY), has been identified as feedback

inhibitors of the RAS/MAPK/ERK signalling cascade triggered by RTKs. Consistent

with their inhibitory involvement in the RTK-stimulated ERK pathway, SPRY is a

putative tumor suppressor, and cells lacking either SPRY expression or function may be

hypersensitive to mitogenic and metastatic signals. SPRY isoforms have been found to be

downregulated in most malignancies studied, including breast (151), prostate (152, 153,

156, 161), liver (155) and lung (160) cancers. Functionally, SPRY has been reported to

interfere in steps of tumorigenesis, including proliferation (151, 155, 160), migration

(148, 160, 161), invasion (148), and cell cycle (148) as well as in vitro (148) and in vivo

(151) tumorigenic potential and formation of metastases (162). The prognostic value

associated with SPRY levels have been established in renal (157), liver (158) and prostate

(153) cancer patients. Moreover, SPRY4 mRNA level serves as a reliable response

marker to Gleevec treatment for patients with gastrointestinal stromal tumors (150).

However, the mechanism by which SPRY is silenced appears to vary depending on the

cancer type (151-153, 155, 156, 160, 161), and contradicting data exist for similar

malignancies (153, 155, 156, 158).

The present study aimed to investigate, in addition to any SPRY downregulation, the

underlying mechanisms and functions of SPRY in ovarian cancer. We demonstrate that

SPRY2 mRNA is downregulated in both biopsies and permanent cell lines derived from

EOC. Furthermore SPRY2 gene is deleted in some of the ovarian tumors and is possibly a

cause of the reduced SPRY2 mRNA. We further demonstrate a positive correlation

between SPRY2 and E-cadherin. Furthermore, the re-introduction of SPRY2 in ovarian

cancer cells partially restored E-cadherin expression levels suppressed by EGF and

antagonized the effect of EGF-induced invasion.

2.2 Materials and methods

  • 2.2.1 The Human Exonic Evidence-Based Oligonucleotide microarray (HEEBO)

The HEEBO microarray (Stanford, CA, USA) employed for the study included

44,544 70-mer probes that were designed using a transcriptome-based annotation of

exonic structure for genomic loci. Pooled RNA from 10 human cancer cell lines of

different origins (Stratagene, Universal Human Reference RNA, Cat 740000) for broad

gene coverage on the array was included as a reference. We examined the mRNA

expression profiles of a series of ovarian tumors from the Vancouver General Hospital

tumor bank obtained from patients who were undergoing surgery during 2004 and 2005.

These cases included the following subtypes: high-grade serous (n = 35), low-grade

serous (n = 2), endometrioid (n = 7), clear cell (n = 3), serous borderline (n = 1),

endometrioid borderline tumor (n = 1) and normal Fallopian tube (n = 1). Approval for

the study was obtained from the University of British Columbia Research Ethics Board

(#H04-60102), and written informed consent was obtained from all participants involved

in the study. HEEBO was performed as described previously (243).

  • 2.2.2 Molecular inversion probe (MIP) copy number analysis

To determine whether deletion of the SPRY2 and SPRY4 genes occurs in human

ovarian tumors, samples from another cohort of patients were utilised to perform MIP

copy number analysis. All women undergoing primary debulking surgery at the

Vancouver General Hospital and British Columbia Cancer Agency in Vancouver,

Canada, between January 2004 and September 2005 were invited, except those with

mucinous and borderline tumors or who had received pre-operative chemotherapy. The

pathology data were reviewed by a pathologist (CBG). The classification and grading of

tumors were performed as described previously (244). We included 28 high-grade serous,

5 high-grade serous/undifferentiated, 3 high-grade undifferentiated, 5 endometrioid, 4

clear cell and 1 low-grade serous tumors. Ethical approval was obtained from the

University of British Columbia Research Ethics Board (#H02-61375 and #H03-70606),

and written informed consent was obtained from all participants involved in the study.

The MIP copy number assay and copy number estimation were performed as described

previously (245). Copy numbers over 3.0 were considered amplification events and copy

numbers below 1.5 were considered deletion events.

  • 2.2.3 The Cancer Genome Atlas (TCGA)

To obtain direct evidence to support a dependency of reduced SPRY2 mRNA

level on gene deletion, we retrieved data from the TCGA database portal

(http://cancergenome.nih.gov/) in September 2011. Copy number data for 585 ovarian

serous cystadenocarcinomas and 587 normal samples (569 matched and 18 unmatched),

as well as data for the gene expression profile of 584 tumors and 18 unmatched normal

samples, were extracted.

  • 2.2.4 Cell culture and reagents

Four non-tumorigenic SV40 Tag-immortalised OSE-derived lines, IOSE-29, IOSE-

80, IOSE-120, and IOSE-398, were generous gifts from Dr. Nelly Auersperg (University

of British Columbia) (246). The human ovarian adenocarcinoma cell line, BG-1, was

kindly provided by Dr. K.S. Korach (National Institute of Environmental Health Sciences,

NIH, Research Triangle Park, NC) (247). CaOV3, OVCAR3 and SKOV3 ovarian cancer

cell lines were obtained from American Type Culture Collection (Manassas, VA). The

cell lines were cultured in MCDB 105 / M199 (1:1), supplemented with 10% heat-

inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 g/ml streptomycin.

The cells were cultured at 37°C and 5% CO 2 . Fetal bovine serum was purchased from

Hyclone Laboratories (Logan, UT). Human recombinant EGF and other tissue culture

materials were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise

stated. Human SPRY2 overexpression constructs (FLAG-SPRY2) and the empty pXJ40-

FLAG vector, which were transfected using Lipofectamine™ 2000 (Invitrogen, Carlsbad,

CA), were gifts from Dr. Graeme R. Guy (the Institute of Molecular and Cell Biology,

Singapore) (143).

  • 2.2.5 Real-time PCR

Real-time PCR was performed using an ABI 7300 real-time thermal cycler (ABI,

Hercules, CA). SPRY2, SPRY4 and the internal control, GAPDH, were amplified in

duplicate with the following PCR primers: SPRY2, forward 5’-

CCCCTCTGTCCAGATCCATA-3’ and reverse 5’-CCCAAATCTTCCTTGCTCAG-3’;

SPRY4, forward 5’-AGCCTGTATTGAGCGGTTTG-3’ and reverse 5’-

GGTCAATGGGTAGGATGGTG-3’, and GAPDH, forward 5’-

GAGTCAACGGATTTGGTCGT-3’ and reverse 5’-GACAAGCTTCCCGTTCTCAG-3’.

  • 2.2.6 Antibodies

Specific antibodies were used to detect proteins via Western blot analysis: the

anti-E-cadherin antibody was obtained from BD Transduction Laboratories (Lexington,

KY), the anti-SPRY2 antibody was purchased from Sigma and the anti-β-actin antibody

was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

  • 2.2.7 Invasion assay

Twenty-four-well transwell filters with an 8-µm pore coated with 1 mg/ml Matrigel

(50 µl/well; BD Sciences, Mississauga, ON, Canada) were used to assess cell invasion.

SKOV3 cells transfected with either control or SPRY2-overexpression constructs were

trypsinized and resuspended in 0.1% FBS medium, with or without EGF, and then seeded

in triplicate in the upper chamber. Next, 1% FBS medium was added to the lower wells.

The chambers were incubated for 24 h at 37°C in a 5% CO 2 atmosphere. Cells that did

not penetrate the filter were removed, and invaded cells on the lower surface of the filter

were fixed with ice-cold methanol, stained with Hoechst 33258, and counted by

epifluorescence microscopy using Northern Eclipse 6.0 software (Empix Imaging,

Mississauga, ON, Canada). Triplicate inserts were used for each individual experiment,

and the results are presented as the mean values.

  • 2.2.8 Statistical analysis

Statistical analysis was performed using Prism Graphing software. Differential

variations in SPRY mRNA levels among ovarian tumor subtypes were assessed using the

Kruskal-Wallis rank test followed by Student’s t test comparing each tumor subtype. The

relative quantification of mRNA expression levels, as assessed by real-time PCR, was

calculated using the 2 ΔΔ Ct method. For the invasion assay and the comparison of E-

cadherin expression levels with the control (using SPRY2-overexpressing cells), a one-

way ANOVA and nonparametric Column Analysis was performed followed by Tukey’s

Multiple Comparison Test to compare all pairs of columns. Columns are not denoted by

the same letter are statistically different (P < 0.05). Data are presented as the mean ± SD

of three or four independent experiments. The Pearson correlation coefficient (r) and

associated probability (P) were calculated when comparing E-cadherin and SPRY2

protein levels using the Spearman nonparametric correlation.

2.3 Results

2.3.1 Levels of SPRY mRNA in ovarian tumors of different pathological subtypes

To examine whether SPRY mRNA were downregulated in ovarian tumors, as is

reported for other malignancies, we compared EOC samples of various histopathological

types, including serous (high-grade, low-grade or borderline), endometrioid (carcinoma

or borderline tumor), clear cell and normal Fallopian tube. SPRY1, SPRY2 and SPRY4

mRNA were included in our array. Significant differences in the SPRY2 mRNA levels

were observed in various tumors types (Kruskal-Wallis test, P = 0.0091, Fig. 2.1). The

lowest mean level of SPRY2 mRNA was observed in the clear cell sample followed by

the high-grade serous carcinomas. All other samples displayed higher mean SPRY2

mRNA levels compared with the reference level. To further assess the variations in

SPRY2 mRNA levels among the categories, we extended our study to perform

comparisons between each subtype using Student's t test. The difference in SPRY2

mRNA levels between high-grade and low-grade serous was statistically significant (P =

0.022, Table 2.1). When comparing pathological subtypes, the mean SPRY2 mRNA

levels in serous and clear cell carcinomas were statistically lower than that of

endometrioid carcinomas (serous vs. endometrioid, P = 0.0007; clear cell vs.

endometrioid, P = 0.022). SPRY4 mRNA levels were not significantly different between

the various subtypes (P = 0.33, data not shown); however, the mean expression level in

high-grade serous was lower than that in the reference and was the lowest among all the

specimens. We utilised three oligonucleotides in the array that corresponded to different

SPRY1 transcripts, for which the expression levels were similar for each subtype and

therefore did not display significant differences (P = 0.396, 0.706 and 0.813, data not

shown).

  • 2.3.2 Levels of SPRY mRNA in immortalised ovarian surface epithelium (IOSE) and

EOC-derived cell lines

Next, we extended our expression analysis to include ovarian cancer cells cultured in

vitro. To enhance data reliability, 4 IOSE cell lines established from individual patients

were included as references for the comparison of SPRY mRNA levels in ovarian cancer

cell lines. Using real-time PCR, we confirmed the observation of reduced SPRY2 mRNA

(Fig. 2.2A) expression in most (3 out of 4) ovarian cancer cell lines tested (BG-1,

OVCAR3 and SKOV3). In addition, the SPRY4 mRNA levels observed in all 4 cancer

cell lines were consistently lower than those of the