AN INVESTIGATION INTO THE ROLE OF THE GHRELIN AXIS IN...

AN INVESTIGATION INTO THE ROLE OF THE GHRELIN

AXIS IN HORMONE-DEPENDENT CANCER AND

CHARACTERISATION OF A NOVEL EXON 3-DELETED

PREPROGHRELIN ISOFORM AND ITS MURINE

HOMOLOGUE

Penelope Lorrelle Jeffery BAppSc (Hons)

Science Research Centre

School of Life Sciences

Queensland University of Technology

A thesis submitted for the degree of Doctor of Philosophy of the

Queensland University of Technology

2005

iii

Abstract

Ghrelin is a 28 amino acid peptide hormone with a unique octanoic acid

modification that has an extensive range of physiological effects, including

stimulation of growth hormone (GH) release, appetite regulation, and modulation of

reproductive functions. The cognate receptor for ghrelin is the growth hormone

secretagogue receptor (GHS-R), a G protein-coupled receptor with two documented

isoforms, the functional GHS-R type 1a and the C-terminally truncated GHS-R type

1b. Several ghrelin variants have also been identified in addition to the n-

octanoylated form of ghrelin. In our laboratory, we have identified a novel exon 3-

deleted preproghrelin variant that retains sequence for the mature ghrelin hormone

and also encodes a novel C-terminal peptide (designated as C-terminal 3 peptide).

There is emerging evidence to suggest that the ghrelin axis, encompassing ghrelin,

several ghrelin variants and both forms of the GHS-R, is implicated in tumour

growth. The objective of this project is to investigate the role of the ghrelin axis in

hormone-dependent cancer and to further characterise the expression and function of

the novel exon 3-deleted preproghrelin isoform.

Hormone-dependent cancers, including prostate and breast cancers, are

significant causes of morbidity and mortality in the Western world. Improved

diagnoses and treatments earlier in the progression of the disease are urgently

required to improve patient outcomes. Growth factors play an integral role in

prostate and breast cancer, particularly in the emergence of aggressive, hormone-

refractory disease that is resistant to standard therapies. We have previously

identified ghrelin as being a novel growth factor for prostate cancer cells in vitro and

have hypothesised that this may be extended to other hormone-dependent cancer

types including breast cancer.

In the current study, techniques including real-time quantitative RT-PCR,

Western blot analysis and immunohistochemistry have been used to determine and

quantitate ghrelin, exon 3-deleted preproghrelin and GHS-R expression in prostate

and breast cancer. Ghrelin and exon 3-deleted preproghrelin are highly expressed in

prostate cancer tissues compared to expression levels in normal prostate glands.

Similarly, breast carcinoma specimens display greater immunoreactivity for ghrelin

iv

and exon 3-deleted preproghrelin than normal breast tissues. Expression of the exon

3-deleted preproghrelin mRNA isoform is upregulated in the oestrogen-independent,

highly malignant MDA-MB-435 breast cancer cell line compared to the non-

tumourigenic MCF-10A breast epithelial cell line, suggesting that augmented

transcription of the isoform is associated with an increased malignant potential in

breast cancer. The functional GHS-R type 1a is expressed in normal breast tissue and

breast cancer specimens and cell lines. In contrast, the truncated GHS-R type 1b

isoform is exclusively expressed in breast carcinoma. These data suggest that GHS-R

type 1b, ghrelin and exon 3-deleted preproghrelin display potential as novel

diagnostic markers for prostate and breast cancer.

These studies have been the first to demonstrate that ghrelin may have an

important role in cell proliferation in breast and prostate cancer. Functional assays

demonstrated that (10nM) ghrelin stimulated proliferation in the LNCaP prostate

cancer cell lines (45.0 ± 1.7% above control, P <0.01) and rapidly activated the ERK

1/2 mitogen-activated kinase (MAPK) pathway in both PC3 and LNCaP cell lines. It

does not, however, protect these cells from chemically-induced apoptosis. The

MAPK inhibitors PD98059 and U0126 blocked ghrelin-induced MAPK activation,

as well as cell proliferation, in both cell lines. Prostate cancer cells secrete mature

ghrelin in vitro, and may therefore stimulate MAPK pathways in an autocrine

manner. Ghrelin also appears to act as a growth factor in breast cancer cell

proliferation, as the growth of MDA-MB-435 and MDA-MB-231 breast cancer cell

lines is significantly increased by ghrelin treatment. Our findings suggest that the

ghrelin axis could provide an important new target for adjunctive therapies for both

breast and prostate cancer.

The C-terminal 3 peptide derived from exon 3-deleted preproghrelin

may be an important new component of the ghrelin axis and studies into its function

are currently in progress. Although it did not induce MAPK cascades or stimulate

proliferation in prostate or breast cancer cell lines, the discovery of a murine

counterpart, exon 4-deleted preproghrelin, indicates that it is highly conserved. Exon

4-deleted preproghrelin is expressed in all mouse tissues examined, with stomach

being the predominant site of synthesis. Other components of the ghrelin axis were

also found to be present in a wide-range of mouse tissues including brain, ovary and

v

prostate. This comprehensive report has paved the way for future work with in vivo

mouse models of cancer.

This study has provided a substantial basis for the further evaluation of

ghrelin, exon 3-deleted preproghrelin and the GHS-R type 1b as novel

diagnostic/prognostic markers for prostate and breast cancer and supports the

rationale for targeting the ghrelin axis for treatment of these tumours.

Keywords: Ghrelin, exon 3-deleted preproghrelin, GHS-R, growth factors, MAPK,

ERK 1/2, hormone-dependent cancer, prostate, breast, diagnostic/prognostic marker,

therapeutic targets.

vi

List of publications, conference abstracts and patents The following is a list of publications and submitted manuscripts, conference

abstracts and patents that have been derived from the work performed for this thesis:

Jeffery PL, Herington AC, Chopin LK (2003) The potential autocrine/paracrine

roles of ghrelin and its receptor in hormone-dependent cancer. Cytokine and Growth

Factor Reviews 14: 113-122.

Jeffery PL, Duncan RP, Yeh AH, Jaskolski RJ, Hammond DS, Herington AC,

Chopin LK (2005) Expression of the ghrelin/GHS-R axis in the mouse: an exon 4-

deleted proghrelin variant encondes a novel C-terminal peptide. Endocrinology 146:

432-440.

Jeffery PL, Murray RE, Yeh AH, McNamara JF, Duncan RP, Francis G, Herington

AC, Chopin LK (2005) Expression and function of the ghrelin axis, including a novel

preproghrelin isoform, in human breast cancer tissues and cell lines. Endocrine

Related Cancer, in press.

Yeh AH*, Jeffery PL*, Duncan RP, Herington AC, Chopin LK (2005) Ghrelin and

a novel preproghrelin isoform are highly expressed in prostate cancer compared to

normal prostate tissue and ghrelin-mediated prostate cancer cell line proliferation

involves the MAPK pathway. Clinical Cancer Research, in press.

* These two authors contributed equally to the work as first authors

Other related publications:

Jeffery PL, Herington AC, Chopin LK (2002) Expression and action of the growth

hormone releasing peptide ghrelin and its receptor in prostate cancer cells lines.

Journal of Endocrinology 172: 7-11.

vii

Gaytan F, Morales C, Barreiro ML, Jeffery PL, Chopin LK, Herington AC,

Casanueva FF, Aguilar E, Dieguez C, Tena-Sempere M (2005) Expression of

Growth Hormone Secretagogue Receptor Type 1a, the Functional Ghrelin Receptor,

in Human Ovarian Surface Epithelium, Mullerian Duct Derivatives and Ovarian

Tumors. Endocrinology 90: 1798-1803.

Conference Abstracts

PL Jeffery, RP Duncan, AH Yeh, RA Jaskolski, DS Hammond, AC Herington, LK

Chopin (2004) Expression of the ghrelin axis in the mouse: an exon 4-deleted

proghrelin variant encodes a novel C terminal peptide. Second International GH-IGF

Symposium, Cairns, Queensland, April 2004.

LK Chopin, AH Yeh, PL Jeffery, AC Herington (2004) Ghrelin promotes cellular

proliferation in the PC3 and LNCaP prostate cancer cell lines through the activation

of mitogen activated protein kinase pathway. Second International GH-IGF

Symposium, Cairns, Queensland, April 2004.

PL Jeffery, AH Yeh, JF McNamara, AC Herington and LK Chopin (2003) The roles

of ghrelin, its cognate receptor and a novel proghrelin isoform in hormone-dependent

cancer and reproduction. Annual meeting of the Society for Endocrinology, Royal

College of Physicians, London, November 2003.

R Jaskolski, RP Duncan, PL Jeffery, C Craven, AC Herington and LK Chopin

(2003) The expression of ghrelin, an exon 4 deleted proghrelin isoform and the

ghrelin receptor in the mouse embryo. ASMR annual meeting, Adelaide, November

2003.

PL Jeffery, AH Yeh, AC Herington and LK Chopin (2003) The autocrine/paracrine

roles of ghrelin and the GHS-R in hormone-dependent cancer. Endocrine Society of

Australia, Novartis junior investigator finalist presentation, Melbourne, September

2003.

viii

LK Chopin, PL Jeffery, JF McNamara, AC Herington (2002) Expression of

components of the ghrelin/Growth hormone secretagogue receptor axis in breast

cancer cell lines and in breast cancer histopathological specimens. US Endocrine

Society 84th Annual meeting, San Francisco, June 2002.

Lisa K Chopin, Penny L. Jeffery, John F. McNamara, Carolyn S. Chan, Ying Dong,

Judith A Clements, Adrian C Herington (2002) Ghrelin and growth hormone

secretagogue receptor (GHS-R) expression in ovarian and endometrial cell lines and

tumours. US Endocrine Society 84th Annual meeting, San Francisco, June 2002.

PL Jeffery, John F. McNamara, Adrian C Herington and LK Chopin (2002) The

identification and expression pattern of a ghrelin variant that encodes a novel peptide

in the human and mouse. Endocrine Society of Australia, annual scientific meeting,

Adelaide, September 2002.

LK Chopin, PL Jeffery, JF McNamara, AC Herington (2002) Expression of the

ghrelin/ growth hormone secretagogue receptor axis in the mouse. GH and IGF

Society Joint Symposium, Boston, October 2002.

LK Chopin, PL Jeffery, JF McNamara, AC Herington (2002) The identification and

expression pattern of a ghrelin mRNA variant encoding a novel peptide in the

human. GH and IGF Society joint Symposium, Boston, October 2002.

Patents

International Patent PCT/AU02/00582

‘Reproductive cancer diagnosis and therapy’

Chopin LK, Jeffery PL, Herington AC (14/11/2002)

ix

Table of Contents pg

Title page i

Abstract and keywords iii

List of publications, conference abstracts and patents vi

Table of Contents ix

Declaration xi

Acknowledgements and dedication xii

List of abbreviations xiii

Chapter 1 – Introduction 1

Chapter 2 – Literature Review 11

Statement of joint authorship 12

Abstract 13

2.1 Introduction 14

2.1.1 The growth hormone secretagogues (GHS) 14

2.1.2 The growth hormone secretagogue receptor (GHS-R) 15

2.1.3 Splice variants of the GHS-R 15

2.1.4 Ghrelin 17

2.1.5 Ghrelin variants 18

2.1.6 Tissue distribution of ghrelin and the GHS-R 21

2.2 The GHRH/GH/IGF axis in cancer 24

2.2.1 GH and insulin-like growth factors (IGFs) 24

2.2.2 GH releasing hormone (GHRH) 26

2.2.3 Ghrelin and the GHS-R in cancer 27

2.3 References 34

Chapter 2B – Literature Review Update 47

2B.1 Ghrelin/GHS-R expression in Cancer 48

2B.2 Interaction of the ghrelin axis with sex steroid hormones 48

2B.3 Functional studies of the role of ghrelin/GHS-R in cancer 50

2B.4 References 52

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Chapter 3 - Ghrelin and a novel preproghrelin isoform are highly expressed in

prostate cancer compared to normal prostate tissue and ghrelin-mediated

prostate cancer cell line proliferation involves the MAPK pathway 59


Abstract 62

Introduction 63

Materials and methods 64

Results 69

Discussion 79

References 88

Chapter 4 - Expression and function of the ghrelin axis, including a novel

preproghrelin isoform, in human breast cancer tissues and cell lines 95


Abstract 98

Introduction 99


Results 105

Discussion 116

References 120

Chapter 5 - Expression of the ghrelin axis in the mouse: an exon 4-deleted

proghrelin variant encodes a novel C terminal peptide 127


Abstract 130

Introduction 131


Results 136

Discussion 153

References 158

Chapter 6 – General Discussion 163

xi

Declaration

The work contained in this thesis has not been previously submitted for a

degree or diploma at any other higher education institution. To the best of my

knowledge and belief, the thesis contains no material previously published or written

by another person except where due references are made.

Signed:…………………………

Penelope Jeffery

Date:……………………………

xii

Acknowledgements and Dedication

I would like to thank my supervisors, Dr Lisa Chopin and Professor

Adrian Herington for their invaluable support, encouragement and friendship during

my candidature. Many thanks also go to all of my colleagues in the Hormone-

dependent cancer program for their advice and friendship. I would like to

acknowledge the financial support I have received from the Australian Postgraduate

Award PhD scholarship scheme and from the QUT Dean of Science top-up

scholarship.

Many thanks and love to my parents, Adrienne and Ross, and to my

partner John, for their boundless patience, support and babysitting and to my

daughter Cordelia, my greatest achievement of all.

This thesis is dedicated to my brother, Randall.

xiii

List of Abbreviations

aa Amino acid

AEBSF 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochoride

AGRF Australian Genome Research Facility

AMV Avian myeloblastosis virus

ANOVA Analysis of variance

ATCC American type culture collection

bp Base pairs

BSA Bovine serum albumin

cDNA Complementary deoxyribose nucleic acid

DAPI 4,6-Diamidino-2-phenylindole

DMEM Dulbecco’s modified eagle medium

DMSO Dimethyl Sulfoxide

DNA Deoxyribose nucleic acid

dNTP Deoxy-nucleotide triphosphate

DTT Dithiothreitol

EDTA Ethylenediamine tetra acetic acid di-sodium salt

EGF Epidermal growth factor

ERK Extracellular-signal regulated kinase

EST Expressed sequenced tag

FBS Foetal bovine serum

FCS Foetal calf serum

g Grams

GGDT Ghrelin gene-derived transcript

GH Growth hormone

GH-R Growth hormone receptor

GHS Growth hormone secretagogue

GHS-R Growth hormone secretagogue receptor

h Hour

HRP Horse radish peroxidase

IGF Insulin-like growth factor

kDa Kilodaltons

xiv

M Molarity (when refering to concentration)

MAPK Mitogen activated protein kinase

mg/ml Milligrams per millilitres

min Minute

ml Millilitre

mM Millimolar

mmol/L Millimoles per litre

mRNA Messenger ribose nucleic acid

MTT 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

mV Millivolts

MW Molecular weight marker

NCBI National centre for biotechnology information

PAGE Polyacrylamide gel electrophoresis

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PKC Protein kinase C

PLC Phospholipase C

pmol Picomole

PSA Prostate specific antigen

RNA Ribose nucleic acid

RPM Revolutions per minute

RPMI Roswell park memorial institute

RT-PCR Reverse transcriptase polymerase chain reaction

s Seconds

SDS Sodium dodecyl sulphate

SEM Standard error of the mean

Ser-3 3rd residue (serine)

SSC Tri-sodium citrate

Ta Annealing temperature

TBS Tris buffered saline

TBST Tris buffered saline/tween

TMD Transmembrane domain

U/ml Units per millilitre

xv

WST-1 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-

tetrazolium monosodium salt

w/v Weight per volume

μg Micrograms

μg/ml Micrograms per millilitre

μl Microlitre

μm Micrometre

μM Micromolar

1

CHAPTER ONE

INTRODUCTION

Description of scientific problem investigated

Hormone-dependent cancer has a significant impact upon morbidity and

mortality rates in the Western world. Extensive research is underway to identify new

therapeutic targets and markers to treat and more effectively diagnose the wide-

ranging spectrum of these cancers that include prostate, breast, endometrial and

ovarian cancers. The most prevalent forms of hormone-dependent cancer, prostate

and breast cancer, account for over 30% of newly diagnosed cancers in Western men

and women respectively (Jemal et al. 2005). Prostate cancer is the second-leading

cause of cancer mortality in men, with males from developed countries facing a

lifetime risk of 1 in 6 of developing prostate cancer (Jemal et al. 2005). Prostate

tumourigenesis is driven largely by steroid androgens and anti-androgen therapy is

the mainstay of prostate cancer treatment (So et al. 2003, Van der Poel, 2004). There

is now considerable evidence that up-regulated growth factor activity promotes an

aggressive, androgen-independent prostate cancer phenotype that is resistant to

traditional hormonal therapies and is associated with a poor prognosis (Feldman and

Feldman, 2001). This highlights the need for adjunctive therapies that can prevent, or

overcome, hormone-refractory disease. The activation of mitogen-activated protein

kinase (MAPK) cascades by growth factors can enhance prostate cancer cell

proliferation and survival (Maroni et al. 2004). Manipulation of MAPK signal

transduction pathways is therefore a novel therapeutic strategy that has been

proposed for the treatment of advanced prostate cancer (Sebolt-Leopold and Herrera,

2004). Diagnosis of prostate cancer has proven problematic due to the non-

specificity of the most widely used tumour marker, serum prostate specific antigen

(PSA) (Thompson et al. 2004). The PSA detection test is also unable to accurately

predict pathological stage and new prognostic markers are clearly required to give a

better profile of prostate tumour grade and severity (Watson and Schalken, 2004).

2

Breast cancer in women and prostate cancer in men share many common

features including epidemiology, mortality rates and hormonal-dependence (Lopez-

Otin and Diamandis, 1998). The effectiveness of selective oestrogen receptor

modulators (SERMs) attests to the importance of oestrogen-withdrawal in the

treatment of breast cancer. As is the case for prostate cancer, however, elevated

growth factor signalling may allow for the emergence of hormone-refractory breast

cancer which is associated with higher mortality rates, particularly in premenopausal

women (Nicholson et al. 2004). Pharmacological molecules designed to block

various growth factors and their receptors have displayed some success in the

laboratory and the clinic, and this field of cancer treatment, including small molecule

therapy, remains promising (Guillemard and Saragovi, 2004). The identification of

new therapeutic targets as well as novel diagnostic and prognostic markers,

particularly in hormone-refractory cancer, is imperative. Growth factors have the

potential to fulfil these roles in the future.

Growth hormone (GH) and its downstream mediators, the insulin-like growth

factors (IGFs), have been implicated in both direct and indirect modulation of

prostate and breast cancer cell function (Chopin and Herington, 1999, Laban et al.

2003). Components of the GH axis have therefore been presented as potential targets

for anti-tumour therapy. In 1999, a 28 amino acid peptide hormone capable of

potently stimulating GH release was purified from stomach tissue (Kojima et al.

1999). This hormone, named ghrelin (ghre: grow), was identified several years after

its endogenous receptor, the growth hormone secretagogue receptor (GHS-R)

(Howard et al. 1996). It is now widely accepted that ghrelin and its receptor are

important new components of the GH axis. Ghrelin action is not restricted to GH

release, however, and it possesses many potentially important physiological

functions including appetite stimulation and energy homeostasis (Nakazato et al.

2001, Shintani et al. 2001), embryo implantation (Tanaka et al. 2003, Duncan et al.

2005) and cardiovascular actions (Benso et al. 2004). Several ghrelin isoforms have

also been identified, including a novel exon 3-deleted preproghrelin variant

discovered in our laboratory (Jeffery et al. 2002). Expression and function of this

variant, along with that of its murine homologue, exon 4-deleted preproghrelin, was

investigated over the course of this project. Ghrelin has also been shown to be a

mitogen for cancer cell lines and our study of the proliferative effect of ghrelin on

3

prostate cancer cells was one of the first published in this field (Jeffery et al. 2002).

The physiological role of ghrelin as a growth factor in hormone-dependent cancer

requires further elucidation.

Overall objectives of the study

Preliminary data from our laboratory suggested that components of the

ghrelin axis display potential as novel therapeutic targets and diagnostic markers for

prostate cancer (Jeffery et al. 2002). The overall objective of this study was to

investigate the role that the ghrelin axis, including the novel exon 3-deleted

preproghrelin isoform, may play in prostate cancer, and to extend these studies to

encompass breast cancer. This study also aimed to provide a better understanding of

the physiology of the human ghrelin axis in general. Expression of the murine

ghrelin axis was also investigated to prepare for the development of future mouse

models.

Specific aims of the study were:

• To investigate the expression of mRNA and protein for ghrelin,

preproghrelin isoforms and the GHS-R in prostate and breast cancer cell

lines and tissues, as well as in normal tissues, using RT-PCR, real time

quantitative RT-PCR, immunohistochemistry and Western blot analysis

• To perform functional assays on prostate and breast cancer cell lines in

vitro treated with synthetic ghrelin

Proliferation, migration and apoptosis assays were performed and the

concentration of GH secreted into the conditioned media measured. The

signalling mechanisms underlying ghrelin action in prostate cancer cells were

also investigated in the laboratory, by studying the involvement of the

mitogen-activated protein kinase (MAPK) pathways.

• To investigate the expression of a mouse homologue of ghrelin and

preproghrelin isoforms in a wide range of mouse tissues using RT-PCR,

real time quantitative RT-PCR and immunohistochemistry

4

Summary of scientific progress linking the journal manuscripts

This thesis is being presented “By Publication” based on Queensland

University of Technology guidelines. Accordingly, each chapter represents a

manuscript that has been published, or has been submitted for publication, in an

international refereed journal.

Chapter 2: Literature Review: The potential autocrine/paracrine roles of ghrelin and

its receptor in hormone-dependent cancer

This literature review was the first to be published regarding the role of

the ghrelin axis in hormone-dependent cancer. This paper reviews the literature

describing the expression, regulation and function of ghrelin and the GHS-R in

cancer, as well as providing an updated assessment of studies exploring the role of

GH and IGF-1 in cancer. The exon 3-deleted preproghrelin isoform was first

discovered in prostate cancer cell lines (Jeffery et al. 2002), and this review contains

a further analysis and schematic depiction of the exon deletion and putative protein

sequence of this isoform. This paper was published in Cytokine and Growth Factor

Reviews (2003) 14: 113-22. A brief literature update, encompassing recent studies on

the ghrelin axis in cancer (2002-2005), is also included at the end of this chapter

(Chapter 2, part B).

Chapter 3: Research article: Ghrelin and a novel preproghrelin isoform are highly

expressed in prostate cancer compared to normal prostate tissue and ghrelin-

mediated prostate cancer cell proliferation involves the MAPK pathway

As a natural progression following our studies in prostate cancer cell lines

(Jeffery et al. 2002) we investigated the signalling pathways underlying ghrelin-

stimulated prostate cancer cell proliferation. This paper contains novel data, derived

from studies performed with the co-author Anthony Yeh, demonstrating that ghrelin

increases prostate cancer cell proliferation via the MAPK pathway. The expression

and function of the exon 3-deleted preproghrelin isoform discovered in our

laboratory (Jeffery et al. 2002), and wild-type ghrelin, are more fully characterised in

5

prostate histopathological specimens and cell lines. This manuscript has been

accepted for publication in Clinical Cancer Research (2005) and is currently in press.

Chapter 4: Research article: Expression and function of the ghrelin axis, including a

novel preproghrelin isoform, in human breast cancer tissues and cell lines

Breast cancer is the most commonly diagnosed malignancy in women in

the Western world (Jemal et al. 2005), and like prostate cancer in men, is highly

sensitive to the effects of growth factors. We therefore decided to investigate the

role of the ghrelin axis in this hormone-dependent cancer. Ghrelin, exon 3-deleted

preproghrelin, and GHS-R expression was demonstrated at the mRNA and protein

level in breast cancer cell lines and tissues. Negligible expression of the GHS-R type

1b in normal breast tissue compared to expression in breast cancer tissues indicates

that this receptor isoform may possess a unique role in breast cancer and has

potential as a diagnostic marker. Ghrelin treatment elicited proliferation in breast

cancer cell lines, as was previously observed in prostate cancer cell lines. Expression

of the exon 3-deleted preproghrelin mRNA isoform was observed to be upregulated

in breast cancer cell lines compared to expression in a non-tumourigenic breast

epithelial cell line. This manuscript has been accepted for publication in Endocrine

Related Cancer (2005) and is currently in press.

Chapter 5: Research article: Expression of the ghrelin axis in the mouse: an exon 4-

deleted proghrelin mouse variant encodes a novel C-terminal peptide

The data generated from the in vitro studies described in Chapters 3 and 4

has provided a substantial base for future work examining the role of the ghrelin axis

in prostate and breast cancer in vivo. Mouse models will be important tools for this

work, therefore, characterisation of the murine ghrelin axis was undertaken and the

data derived from these studies are presented in Chapter 5. This paper also describes

the expression of a murine homologue of exon 3-deleted preproghrelin and

demonstrates that the novel isoform is conserved across species, implying

physiological significance. The murine ghrelin isoform was shown to be expressed at

the mRNA and protein levels by quantitative RT-PCR, Western blot and

immunohistochemical analysis. This research article was the first comprehensive

6

report on the expression of the ghrelin axis and the ghrelin isoform in the mouse

(Jeffery et al. 2005) and has been published in Endocrinology (2005, 146: 432-40).

7

REFERENCES

Benso A, Broglio F, Marafetti L, Lucatello B, Seardo MA, Granata R, Martina V,

Papotti M, Muccioli G, Ghigo E (2004) Ghrelin and synthetic growth hormone

secretagogues are cardioactive molecules with identities and differences. Seminars

in Vascular Medicine 4: 107-114.

Chopin L, Herington A (1999) The growth hormone insulin-like growth factor axis

and prostate cancer. Clinical Biochemists Reviews 20: 3-16.

Duncan RP, Jaskolski RA, Jeffery PL, Herington AC, Chopin LK (2005) A potential

role for ghrelin in embryo implantation. In preparation.

Feldman BJ, Feldman D (2001) The development of androgen-independent prostate

cancer. Nature Reviews Cancer 1: 34-45.

Guillemard V, Saragovi HU (2004) Novel approaches for targeted cancer therapy.

Current Cancer Drug Targets 4: 313-326.

Howard A, Feighner S, Cully D, Arena J, Liberator P, Rosenblum C, Hamelin M,

Hreniuk D, Palyha O, Anderson J, Paress P, Diaz C, Chou M, Liu K, McKee KK,

Pong S, Chuang L, Elbrecht A, Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji

D, Dean D, Melillo D, Patchett AA, Nargund R, Grifin P, DeMartino J, Gupta S,

Schaefer JA, Smith R, Van der Ploeg L (1996) A receptor in pituitary and

hypothalamus that functions in growth hormone release. Science 12: 137-145.


hormone releasing peptide ghrelin and its receptor in prostate cancer cells lines.




deleted proghrelin variant encodes a novel C-terminal peptide. Endocrinology 146:

432-440.

8

Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, Feuer EJ, Thun MJ

(2005) Cancer Statistics, 2005. CA: A cancer journal for clinicians 55: 10-30.

Kojima M, Hosodo H, Date Y, Nakazato M, Matsuo H, Kanagawa K (1999) Ghrelin

is a growth hormone releasing acylated peptide from stomach. Nature 402: 656-660.

Laban C, Bustin SA, Jenkins PJ (2003) The GH-IGF-I axis and breast cancer. Trends

in Endocrinology and Metabolism 14: 28-34.

Lopez-Otin C, Diamandis EP (1998) Breast and prostate cancer: an analysis of

common epidemiological, genetic and biochemical features. Endocrine reviews 19:

365-396.

Maroni PD, Koul S, Meacham RB, Koul H. (2004) Mitogen activated protein kinase

signal transduction pathways in the prostate. Cell Communication and Signaling 2:5-

18.

Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kengawa K, Matsukura S

(2001) A role for ghrelin in the central regulation of feeding. Nature 409: 194-198.

Nicholson RI, Staka C, Boyns F, Hutcheson IR, Gee JMW (2004) Growth factor-

driven mechanisms associated with resistance to estrogen deprivation in breast

cancer: new opportunities for therapy. Endocrine-Related Cancer 11: 623-641.

Sebolt-Leopold JS, Herrera R (2004) Targeting the mitogen-activated protein kinase

cascade to treat cancer. Nature Reviews Cancer 4: 937-947.

Shintani M, Ogawa Y, Ebihara K, Aizawa-Abe M, Miyanaga F, Takaya K, Hayashi

T, Inoue G, Hosoda K, Kojima M, Kangawa K, Nakao K (2001) Ghrelin, an

endogenous growth hormone secretagogue, is a novel orexigenic peptide that

antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1

receptor pathway. Diabetes 50: 227-232.

9

So AI, Hurtado-Coll A, Gleave ME (2003) Androgens and prostate cancer. Journal

of Urology 21: 325-337.

Tanaka K, Minoura H, Isobe T, Yonaha H, Kawato H, Wang DF, Yoshida T, Kojima

M, Kangawa K, Toyoda N (2003) Ghrelin is involved in the decidualization of

human endometrial stromal cells. Journal of Clinical Endocrinology and Metabolism

88: 2335-2340.

Thompson IM, Pauler DK, Goodman PJ, Tangen CM, Lucia MS, Parnes HL,

Minasian LM, Ford LG, Lippman SM, Crawford ED, Crowley JJ, Coltman CA

(2004) New England Journal of Medicine 350: 2239-2246.

Van de Poel HG (2004) Smart drugs in prostate cancer. European Urology 45: 1-7.

Watson RWG, Schalken JA (2004) Future opportunities for the diagnosis and

treatment of prostate cancer. Prostate Cancer and Prostatic Diseases. 7 (Suppl 1): 8-

13.

11

CHAPTER TWO

LITERATURE REVIEW

THE POTENTIAL AUTOCRINE/PARACRINE ROLES

OF GHRELIN AND ITS RECEPTOR IN

HORMONE-DEPENDENT CANCER

Penny L Jeffery, Adrian C Herington and Lisa K Chopin

Cytokine and Growth Factor Reviews (2003) 14: 113-22.

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This article is not available online. Please consult the hardcopy thesis available from the QUT Library

47

CHAPTER TWO (PART B)

LITERATURE REVIEW UPDATE

48

Literature Review Update

From the time that our review was published in early 2003, the body of

research concerning the role of the ghrelin axis in cancer has expanded considerably;

hence a brief update of the literature is required. This chapter contains a review of

recent articles pertaining to ghrelin and the GHS-R in cancer, as this is the focus of

subsequent chapters in this thesis.

2B.1 Ghrelin/GHS-R expression in cancer

The number of tumour types that have been demonstrated to express

ghrelin and the GHS-R has increased in recent years. Endocrine tumours that

produce and secrete high quantities of ghrelin into the circulation (named

ghrelinomas), have been identified. A grossly elevated serum ghrelin concentration

was measured in a patient with a highly malignant gastric ghrelinoma (Tsolakis et al.

2004) and the occurance of a rare pancreatic ghrelinoma has been reported (Corbetta

et al. 2003). A subset of pancreatic islet cell tumours associated with multiple

endocrine neoplasia-1 (MEN-1) show ghrelin immunoreactivity (Iwakura et al.

2002). Ghrelin and both isoforms of the GHS-R are expressed in a bronchial

neuroendocrine tumor, but not in adjacent normal lung tissue, suggesting a

modulatory role for the ghrelin axis in this tumour type (Arnaldi et al. 2003).

Ghrelin and the functional GHS-R type 1a are also expressed in reproductive system

malignancies including highly differentiated testicular tumours (Gaytan et al. 2004)

and ovarian tumours (Gaytan et al. 2005).

2B.2 Interaction of the ghrelin axis with sex steroid hormones

A plethora of experimental and epidemiological studies has demonstrated

that sex steroids, including oestrogens and androgens, are the prime modulators of

hormone-dependent cancers, particularly those of the breast and prostate (Haslam

and Woodward, 2003, So et al. 2003). Recent research has indicated that ghrelin and

its cognate receptor (GHSR 1a) may interact with sex steroid hormones including

androgens, oestrogens and progesterone. Ovarian thecal and granulosa cells are

prime sites of oestrogen biosynthesis in the pre-menopausal female (Ryan and Petro,

49

1966), and these cell types express ghrelin and the GHSR 1a in a variable pattern

across the normal menstrual cycle, suggesting that ghrelin may modulate oestrogen

function (Gaytan et al. 2003). This may only be relevant to autocrine/paracrine

oestrogen physiology, as ghrelin administration in rats did not perturb serum

estradiol levels (Fernandez-Fernandez et al. 2005), and surprisingly, there are no

available data regarding the impact of ghrelin treatment on oestrogen levels in

humans.

Conversely, oestrogen may regulate ghrelin expression, as in rats

oophorectomy significantly increases plasma ghrelin levels and the number of

ghrelin-immunoreactive cells in the stomach (Matsubara et al. 2004). This effect is

reversed upon administration of 17-β-estradiol indicating that oestrogen negatively

regulates ghrelin production in the rat (Matsubara et al. 2004). In humans studies

however, oestrogen supplementation increased plasma ghrelin concentrations in

severely-undernourished female patients (Grinspoon et al. 2004) and in

postmenopausal women receiving HRT (hormone replacement therapy), although the

mechanisms behind these effects are unknown (Kellokoski et al. 2005). Post-

menopausal HRT use has been associated with an increased risk of developing breast

cancer (Chlebowski et al. 2003), although studies in this field are contradictory

(Creasman, 2005). Oestrogen also potentiates ghrelin-induced pulsatile GH secretion

(Veldhuis and Bowers, 2003), and is an important modulator of GH function (Leung

et al. 2004). GH is emerging as an important oncogenic hormone in breast cancer

(Waters and Conway-Campbell, 2004). No research has been published to date

regarding the potential interaction of ghrelin with oestrogen in the context of breast

cancer.

Other sex hormones appear to influence ghrelin secretion. Androgen

levels in women with polycystic ovary disease are elevated and may inhibit ghrelin

secretion, resulting in lower circulating ghrelin levels compared to normal controls

(Pagotto et al. 2002, Panidis et al. 2005). Ghrelin levels increase to normal

concentrations in these patients after androgen-blockade (Gambineri et al. 2003).

Ghrelin and GHSR 1a co-immunoreactivity is present in ovarian hilus interstitial

cells which are capable of testosterone secretion, under the control of luteinising

hormone (Gaytan et al. 2003, Erickson et al. 1985). This research indicates that

50

ghrelin may regulate androgen secretion and/or function in the ovary (Gaytan et al.

2003). Ghrelin and the GHSR 1a are also co-expressed in the steroidogenic Leydig

cells of the testis and ghrelin inhibits testosterone secretion from testicular tissue in

vitro, potentially by downregulating the expression of key enzymes in the

steroidogenic pathway (Gaytan et al. 2004).

The synthetic progestin molecule, cyproterone acetate, is the most

frequently used steroidal anti-androgen in prostate cancer therapy (Culig et al. 2004).

The recent finding that cyproterone acetate significantly elevates total ghrelin levels

in healthy men (Gambineri et al. 2005) may hold significance for the treatment of

men with advanced prostate cancer, as cell lines derived from metastatic prostate

tumours proliferate in response to ghrelin treatment (Jeffery et al. 2002).

2B.3 Functional studies on the role of ghrelin/GHS-R in cancer

Enhanced proliferation in response to ghrelin treatment has now been

demonstrated in numerous cell lines and many studies have investigated the signal

transduction pathways underlying this response. Exogenous ghrelin increases the

proliferative, invasive and migratory potential of pancreatic adenocarcinoma cells in

vitro, via augmented Akt phosphorylation (Duxbury et al. 2003). Ghrelin activates

the ERK1/2 MAPK pathway and thereby stimulates growth in adrenocortical cells

(Andreis et al. 2003, Mazzocchi et al. 2004), 3T3-L1 adipocytes (Kim et al. 2004)

and the rodent somatotroph pituitary tumour cell line GH3 (Nanzer et al. 2004).

Proliferation of primary cultures of rodent osteoblasts also increases after treatment

with physiological concentrations of ghrelin (Maccarinelli et al. 2005), whilst rodent

neuronal proliferation is stimulated by ghrelin both in vivo and in vitro (Zhang et al.

2004). Endogenous ghrelin may be part of an autocrine loop sustaining growth of

human erythroleukemic (HEL) cells, as blocking antibodies inhibit proliferation of

this cell line in vitro (De Vriese et al. 2005). The growth-promoting role of ghrelin

may also be attributed to its anti-apoptotic actions in many cell lines (Baldanzi et al.

2002, Kim et al. 2004, Mazzocchi et al. 2004, Kim et al. 2005). Apoptosis plays an

integral role in tumour growth; therefore ghrelin may contribute to tumourigenesis by

enhancing tumour cell survival. Some conflicting studies, however, have reported an

inhibitory role for ghrelin in tumour biology. For example, apoptosis is increased in

51

aldosteronoma cell lines after exposure to exogenous ghrelin (Belloni et al. 2004). In

agreement with their previous work in breast cancer cells (Cassoni et al. 2001),

Volante and co-workers have demonstrated that ghrelin inhibits proliferation of

thyroid carcinoma cell lines in vitro, an effect that may be mediated by a receptor

other than the GHS-R (Volante et al. 2003). This research group has also recently

published data that agreed with our own studies (Jeffery et al. 2002) regarding the

mitogenic effect of ghrelin in the PC3 prostate cancer cell line (Cassoni et al. 2004).

The non-acylated form of ghrelin may have a negative impact upon cell growth, as

the des-acyl ghrelin overexpressing transgenic mouse displays a small phenotype and

lowered GH/IGF-I levels (Ariyasu et al. 2005). The physiological effect of ghrelin

on cultured cells in vitro is clearly cell type-specific and may be reliant upon GHS-R

isoform expression, intracellular signalling crosstalk, and ghrelin acylation status;

however, this phenomenon is yet to be fully explored.

52

REFERENCES

Andreis PG, Malendowicz LK, Trejter M, Neri G, Spinazzi R, Rossi GP, Nussdorfer

GG (2003) Ghrelin and growth hormone secretagogue receptor are expressed in the

rat adrenal cortex: Evidence that ghrelin stimulates the growth, but not secretory

activity of adrenal cells. FEBS Letters 536: 173-179.

Ariyasu H, Takaya K, Iwakura H, Hosoda H, Akamizu T, Arai Y, Kangawa K &

Nakao K (2005) Transgenic mice overexpressing des-acyl ghrelin show small

phenotype. Endocrinology 146: 355-364.

Arnaldi G, Mancini T, Kola B, Appolloni G, Freddi S, Concettoni C, Bearzi I,

Masini A, Boscaro M & Mantero F (2003) Cyclical Cushing's syndrome in a patient

with a bronchial neuroendocrine tumour (typical carcinoid) expressing ghrelin and

growth hormone secretagogue receptors. Journal of Clinical Endocrinology and

Metabolism 88: 5834-5840.

Baldanzi G, Filigheddu N, Cutrupi S, Catapano F, Bonissoni S, Fubini A, Malan D,

Baj G, Granata R, Broglio F, Papotti M, Surico N, Bussolino F, Isgaard J, Deghenghi

R, Sinigaglia F, Prat M, Muccioli G, Ghigo E, Graziani A (2002) Ghrelin and des-

acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through

ERK1/2 and PI 3-kinase/AKT. Journal of Cell Biology 159: 1029-1037.

Belloni AS, Macchi C, Rebuffat P, Conconi MT, Malendowicz LK, Parnigotto PP,

Nussdorfer GG (2004) Effect of ghrelin on the apoptotic deletion rate of different

types of cells cultured in vitro. International Journal of Molecular Medicine 14:165-

167.

Cassoni P, Papotti M, Ghe C, Catapano F, Sapino A, Graziani A, Deghenghi R,

Reissmann T, Ghigo E & Muccioli G (2001) Identification, characterisation and

biological activity of specific receptors for natural (ghrelin) and synthetic growth

hormone secretagogues and analogs in human breast carcinoma and cell lines.

Journal of Clinical Endocrinology and Metabolism 86: 1738-1745.

53

Cassoni P, Ghe C, Marrocco T, Tarabra E, Allia E, Catapano F, Deghenghi R, Ghigo

E, Papotti M, Muccioli G (2004) Expression of ghrelin and biological activity of

specific receptors for ghrelin and des-acyl ghrelin in human prostate neoplasms and

related cell lines. European Journal of Endocrinology 150: 173-84.

Chlebowski RT, Hendrix SL, Langer RD, Stefanick ML, Gass M, Lane D,

Rodabough RJ, Gilligan MA, Cyr MG, Thomson CA, Khandekar J, Petrovitch H,

McTiernan A (2003) Influence of estrogen plus progestin on breast cancer and

mammography in healthy postmenopausal women: the Women's Health Initiative

Randomized Trial. Journal of the American Medical Association 289: 3243-3253.

Corbetta S, Peracchi M, Cappiello V, Lania A, Lauri E, Vago L, Beck-Peccoz P,

Spada A (2003) Circulating ghrelin levels in patients with pancreatic and

gastrointestinal neuroendocrine tumours: identification of one pancreatic ghrelinoma.


Creasman WT (2005) Breast cancer: the role of hormone therapy. Seminars in

Reproductive Medicine 23: 167-171.

Culig Z, Bartsch G, Hobisch A (2004) Antiandrogens in prostate cancer endocrine

therapy. Current Cancer Drug Targets 4:455-461.

De Vriese C, Gregoire F, DE Neef P, Robberecht P, Delporte C (2005) Ghrelin is

produced by the human erythroleukemic HEL cell line and involved in an autocrine

pathway leading to cell proliferation. Endocrinology 146: 1514-1522.

Duxbury MS, Waseem T, Ito H, Robinson MK, Zinner MJ, Ashley SW, Whang EE

(2003) Ghrelin promotes pancreatic adenocarcinoma cellular proliferation and

invasiveness. Biochemical and Biophysical Research Communications 309: 464-468.

Erickson GF, Magoffin DA, Dyer CA, Hofeditz C (1985) The ovarian androgen

producing cells: a review of structure/function relationships. Endocrine Reviews 6:

371–339.

54

Fernandez-Fernandez R, Navarro VM, Barreiro ML, Vigo EM, Tovar S, Sirotkin

AV, Casanueva FF, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M (2005) Effects

of chronic hyperghrelinemia on puberty onset and pregnancy outcome in the rat.

Endocrinology 146: 3018-3025.

Gambineri A, Pagotto U, Tschop M, Vicennati V, Manicardi E, Carcello A, Cacciari

M, De Iasio R, Pasquali R (2003) Anti-androgen treatment increases circulating

ghrelin levels in obese women with polycystic ovary syndrome. Journal of

Endocrinological Investigations 26: 629-634.

Gambineri A, Pagotto U, De Lasio R, Meriggiola MC, Costantino A, Patton L, Pelusi

C, Pelusi G, Pasquali R (2005) Short-term modification of sex hormones is

associated with changes in ghrelin circulating levels in healthy normal-weight men.

Journal of Endocrinological Investigations. 28: 241-246.

Gaytan F, Barreiro ML, Chopin LK, Herington AC, Morales C, Pinilla L, Casanueva

FF, Aguilar E, Diéguez C, Tena-Sempere M (2003) Immunolocalization of Ghrelin

and Its Functional Receptor, the Type 1a Growth Hormone Secretagogue Receptor,

in the Cyclic Human Ovary. Journal of Clinical Endocrinology and Metabolism 88:

879-887.

Gaytan F, Barreiro ML, Caminos JE, Chopin LK, Herington AC, Morales C, Pinilla

L, Paniagua R, Nistal M, Casanueva FF, Aguilar E, Dieguez C, Tena-Sempere M

(2004) Expression of ghrelin and its functional receptor, the type 1a growth hormone

secretagogue receptor, in normal human testis and testicular tumors. Journal of

Clinical Endocrinology and Metabolism 89: 400-409.

Gaytan F, Morales C, Barreiro ML, Jeffery P, Chopin LK, Herington AC, Casanueva

FF, Aguilar E, Dieguez C, Tena-Sempere M (2005) Expression of growth hormone

secretagogue receptor type 1a, the functional ghrelin receptor, in human ovarian

surface epithelium, mullerian duct derivatives and ovarian tumors. Endocrinology

90: 1798-1804.

55

Grinspoon S, Miller KK, Herzog DB, Grieco KA, Klibanski A (2004) Effects of

estrogen and recombinant human insulin-like growth factor-I on ghrelin secretion in

severe undernutrition. Journal of Clinical Endocrinology and Metabolism 89: 3988-

3993.

Haslam SZ, Woodward TL (2003) Host microenvironment in breast cancer

development: epithelial-cell-stromal-cell interactions and steroid hormone action in

normal and cancerous mammary gland. Breast Cancer Research 5: 208-215.

Iwakura H, Hosoda K, Doi R, Komoto I, Nishimura H, Son C, Fujikura J, Tomita T,

Takaya K, Ogawa Y, Hayashi T, Inoue G, Akamizu T, Hosoda H, Kojima M,

Kangawa K, Imamura M, Nakao K (2002) Ghrelin expression in islet cell tumours:

augmented expression of ghrelin in a case of glucagonoma with multiple endocrine

neoplasm type I. Journal of Clinical Endocrinology and Metabolism 87: 4885-4888.

Jeffery P, Herington A, Chopin L (2002) Expression and action of the growth

hormone releasing peptide ghrelin and its receptor in prostate cancer cell lines.


Kellokoski E, Poykko SM, Karjalainen AH, Ukkola O, Heikkinen J, Kesaniemi YA,

Horkko S (2005) Estrogen replacement therapy increases plasma ghrelin levels.


Kim MS, Yoon CY, Jang PG, Park YJ, Shin CS, Park HS, Ryu JW, Pak YK, Park

JY, Lee KU, Kim SY, Lee HK, Kim YB, Park KS (2004) The mitogenic and

antiapoptotic actions of ghrelin in 3T3-L1 adipocytes. Molecular Endocrinology 18:

2291-2301.

Kim SW, Her SJ, Park SJ, Kim D, Park KS, Lee HK, Han BH, Kim MS, Shin CS,

Kim SY (2005) Ghrelin stimulates proliferation and differentiation and inhibits

apoptosis in osteoblastic MC3T3-E1 cells. Bone 37: 359-369.

Leung KC, Johannsson G, Leong GM, Ho KK (2004) Estrogen regulation of growth

hormone action. Endocrine Reviews 25: 693-721.

56

Maccarinelli G, Sibilia V, Torsello A, Raimondo F, Pitto M, Giustina A, Netti C,

Cocchi D (2005) Ghrelin regulates proliferation and differentiation of osteoblastic

cells. Journal of Endocrinology 184: 249-256.

Matsubara M, Sakata I, Wada R, Yamazaki M, Inoue K, Sakai T (2004) Estrogen

modulates ghrelin expression in the female rat stomach. Peptides 25: 289-297.

Mazzocchi G, Neri G, Rucinski M, Rebuffat P, Spinazzi R, Malendowicz LK,

Nussdorfer GG (2004) Ghrelin enhances the growth of cultured human adrenal zona

glomerulosa cells by exerting MAPK-mediated proliferogenic and antiapoptotic

effects. Peptides 25: 1269-1277.

Nanzer AM, Khalaf S, Mozid AM, Fowkes RC, Patel MV, Burrin JM, Grossman A,

Korbonits M (2004) Ghrelin exerts a proliferative effect on a rat pituitary

somatotroph cell line via the mitogen-activated protein kinase pathway. European


Pagotto U, Gambineri A, Vicennati V, Heiman ML, Tschop M, Pasquali R (2002)

Plasma ghrelin, obesity, and the polycystic ovary syndrome: correlation with insulin

resistance and androgen levels. Journal of Clinical Endocrinology and Metabolism

87: 5625-5629.

Panidis D, Farmakiotis D, Koliakos G, Rousso D, Kourtis A, Katsikis I, Asteriadis C,

Karayannis V, Diamanti-Kandarakis E (2005) Comparative study of plasma ghrelin

levels in women with polycystic ovary syndrome, in hyperandrogenic women and in

normal controls. Human Reproduction 20: 2127-2132.

Ryan KJ, Petro Z (1966) Steroid biosynthesis by human ovarian granulosa and thecal

cells. Journal of Clinical Endocrinology and Metabolism 26: 46-52.

So AI, Hurtado-Coll A, Gleave ME (2003) Androgens and prostate cancer. Journal

of Urology 21: 325-337.

57

Tsolakis AV, Portela-Gomes GM, Stridsberg M, Grimelius L, Sundin A, Eriksson

BK, Oberg KE, Janson ET (2004) Malignant gastric ghrelinoma with

hyperghrelinemia. Journal of Clinical Endocrinology and Metabolism 89: 3739-

3744.

Veldhuis JD, Bowers CY (2003) Sex steroid modulation of growth hormone (GH)

secretory control: three-peptide ensemble regulation under dual feedback restraint by

GH and IGF-1. Endocrine 22: 25-40.

Volante M, Allia E, Fulcheri E, Cassoni P, Ghigo E, Muccioli G, Papotti M (2003)

Ghrelin in fetal thyroid and follicular tumors and cell lines: expression and effects on

tumor growth. American Journal of Pathology 162: 645-654.

Waters MJ, Conway-Campbell BL (2004) The oncogenic potential of autocrine

human growth hormone in breast cancer. Proceedings of the National Academy of

Sciences of the United States of America 101:14992-14993.

Zhang W, Lin TR, Hu Y, Fan Y, Zhao L, Stuenkel EL, Mulholland MW (2004)

Ghrelin stimulates neurogenesis in the dorsal motor nucleus of the vagus. Journal of

Physiology 559: 729-737.

59

CHAPTER THREE

GHRELIN AND A NOVEL PREPROGHRELIN ISOFORM ARE

HIGHLY EXPRESSED IN PROSTATE CANCER COMPARED

TO NORMAL PROSTATE TISSUE AND GHRELIN-MEDIATED

PROSTATE CANCER CELL LINE PROLIFERATION

INVOLVES THE MAPK PATHWAY

Anthony H. Yeh*, Penelope L. Jeffery*, Russell P. Duncan, Adrian C. Herington and

Lisa K. Chopin

* These two authors contributed equally to the work as first authors.

Clinical Cancer Research (2005), in press

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95

CHAPTER FOUR

EXPRESSION AND FUNCTION OF THE GHRELIN AXIS,

INCLUDING A NOVEL PREPROGHRELIN ISOFORM, IN

HUMAN BREAST CANCER TISSUES AND CELL LINES

Jeffery PL, Murray RE, Yeh AH, McNamara JF, Duncan RP, Francis G,

Herington AC and Chopin LK.

Endocrine-Related Cancer (2005), in press

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127

CHAPTER FIVE

EXPRESSION OF THE GHRELIN AXIS IN THE MOUSE: AN

EXON 4-DELETED MOUSE PROGHRELIN VARIANT

ENCODES A NOVEL C-TERMINAL PEPTIDE

PL Jeffery, RP Duncan, AH Yeh, RA Jaskolski, DS Hammond, AC Herington and

LK Chopin.

Endocrinology (2005) 146: 432-440.

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163

CHAPTER SIX

GENERAL DISCUSSION

The discovery of the peptide hormone ghrelin in 1999, several years after

its endogenous receptor, the growth hormone secretagogue receptor (GHS-R) was

identified, has had a substantial impact upon the field of endocrinology. The

ubiquitous expression of ghrelin in the body, as well as its significant concentration

in serum (Kojima et al. 1999), suggests a wide range of physiological roles for this

unique hormone, in addition to growth hormone (GH) release. There is an expanding

body of research supporting a role for ghrelin and its receptor in cancer. Our previous

work published in 2002, was the first to report the expression of ghrelin, an exon 3-

deleted preproghrelin mRNA variant, and the GHS-R in prostate cancer cell lines and

also reported the growth-promoting effect of ghrelin on PC3 prostate cancer cells in

vitro (Jeffery et al. 2002). This work led to our hypothesis that ghrelin is a growth

factor in prostate cancer and, potentially, other cancers. Prostate and breast cancer

are hormone-dependent cancers that are significant causes of cancer mortality in

Western men and women respectively (Jemal et al. 2005) and are sensitive to the

effects of growth factors and cytokines. Therefore, a logical extension of my initial

studies in prostate cancer cell lines (Jeffery et al. 2002) was to investigate the role of

the ghrelin axis in breast cancer.

Our hypothesis that ghrelin is a growth factor for cancer required further

elucidation and therefore the objectives of this thesis were to investigate the

expression and function of the ghrelin axis in human hormone-dependent cancers,

specifically prostate and breast cancer. These studies include the description of a

novel ghrelin isoform, exon 3-deleted preproghrelin. The salient findings of this

project include the full description of exon 3-deleted preproghrelin expression in

prostate and breast cancer, the identification of ghrelin and exon 3-deleted

preproghrelin as potential diagnostic markers for cancer and the identification of the

ghrelin axis as a novel target for therapeutics. A full characterisation of the murine

ghrelin axis, as a prelude to future in vivo studies using mouse models, has also been

performed.

164

Characterisation of the exon 3-deleted preproghrelin isoform

The expression of the human exon 3-deleted preproghrelin mRNA variant

was first identified in prostate cancer cell lines (Jeffery et al. 2002) and in this thesis

I have characterised its expression and explored the potential functions of this novel

isoform in prostate cancer and breast cancer tissues and cell lines. Translation of the

full-length human preproghrelin transcript results in a 117 amino acid peptide that is

processed to produce the 28 amino acid mature ghrelin hormone (Kojima et al.

1999). Low molecular weight peptides derived from the carboxy terminus of

proghrelin, which may possess cardiovascular activity, are also present in human

serum (Pemberton et al. 2003). Exclusion of exon 3 from the preproghrelin

transcript results in a truncated preproghrelin peptide of 91 amino acids, with a

unique 16 amino acid C-terminal peptide sequence (C terminal 3 peptide). The C-

terminal peptide begins with a pair of arginine residues, known to be a classic

protease cleavage signal for prohormones (Docherty and Steiner, 1982, Seidah and

Chretien, 1997). Bioinformatic analysis of the dibasic sequence demonstrates that it

is a trypsin cleavage site (Expasy Proteomics Server, Swiss Institute of

Bioinformatics), indicating that C- terminal 3 peptide may be cleaved and exist

independently from the rest of exon 3-deleted preproghrelin. Using Western blot

analysis, we were able to determine that cleavage of C terminal 3 peptide from the

rest of the exon 3-deleted preproghrelin peptide does occur in human stomach tissue

and in LNCaP prostate cancer cell lysate (Chapter 3). Importantly, the exon 3-

deleted preproghrelin isoform retains sequence coding for the mature ghrelin peptide

(Figure 1). Using primers specific for the exon 3-deleted preproghrelin transcript, I

have now demonstrated the expression of the isoform in prostate (Chapter 3) and

breast cancer specimens and cell lines (Chapter 4), and in a range of other human

tissues including anterior pituitary and placenta (Jeffery et al. in preparation). The

antibody used to detect protein expression of the exon 3-deleted preproghrelin was

raised against the C-terminal peptide and displayed specificity for the isoform. This

is the only antibody currently available that can distinguish the unique isoform from

full-length preproghrelin protein and has enabled detection of exon 3-deleted

preproghrelin expression in prostate and breast tissue specimens by

immunohistochemistry. Measurement of C-ghrelin peptide concentrations in human

blood has recently been successfully performed (Pemberton et al. 2003) and a similar

165

approach using HPLC coupled with RIA could be used in the future to quantitate C-

terminal 3 peptide immunoreactivity in serum taken from prostate and breast

cancer patients.

A comparison of the exon 3-deleted preproghrelin sequence with those in

EST databases (Blast, NCBI) unearthed an homologous preproghrelin sequence in a

mixed mouse pancreas library (GenBank accession # BM054332), leading to the

discovery and characterisation of the murine homologue (Chapter 5). This isoform

was named exon 4-deleted preproghrelin because the mouse ghrelin gene was known

to consist of 5 exons (Tanaka et al. 2001). It has since been demonstrated that human

ghrelin gene also consists of five exons (Kanamoto et al. 2004), although the human

exon 3-deleted preproghrelin has been named according to coding exons only.

Along with murine GHS-R and ghrelin, exon 4-deleted preproghrelin was detected in

a wide-range of mouse tissues, with stomach being the predominant site of exon 4-

deleted preproghrelin synthesis (Jeffery et al. 2005; Chapter 5). The unique C-

terminal peptide of mouse exon 4-deleted preproghrelin exhibits significant

homology (approximately 60%) with its human counterpart. This peptide sequence

conservation suggests a functional role for the novel isoform.

Exon 3-deleted preproghrelin

Full-length preproghrelin

Signal peptide Ghrelin C-peptide

Amino acids 23 51 117

Signal peptide Ghrelin

RPQPTSDRPQALLTSL (C-terminal 3 peptide)

C-peptideProteolytic cleavage site

Amino acids 23 51 91

Figure 1. Schematic depiction of full-length and exon 3-deleted preproghrelin proteins.

Full-length preproghrelin is a 117 amino acid prohormone which is processed to produce the

mature ghrelin hormone. Deletion of exon 3 from the preproghrelin transcript generates a

premature stop codon and therefore a truncated C-peptide with a novel pro-fragment (C-

terminal 3 peptide). The introduction of a dibasic cleavage site (RR) may allow cleavage of this

C-terminal pro-fragment in vivo.

166

The identification of ghrelin and exon 3-deleted preproghrelin as potential

diagnostic markers for hormone-dependent cancer

Better indicators of prostatic disease and tumour progression are urgently

required. The widely used serum prostate-specific antigen (PSA) test for prostate

malignancy is limited by poor specificity (Thompson et al. 2004), and there are

currently no reliable prognostic markers for prostate cancer (Watson and Schalken,

2004). Similarly, there are few validated prognostic markers for breast cancer,

although expression of growth factors and their receptors (for example HER-2/neu)

are often included in diagnostic profiles of breast disease (Coradini and Daidone,

2004). Immunohistochemistry is commonly employed for detection and localization

of protein expression in histological sections; however, it is also emerging as an

important tool in the diagnosis of malignant changes in tissue, as well as for grading

tumour severity. Prognostication aided by immunohistochemical markers has the

potential to better predict prostate cancer behaviour than traditional histological

grading alone (Sivridis et al. 2002, Epstein, 2004). The immunohistochemical studies

performed in this project revealed that both ghrelin and the novel preproghrelin

isoform are more abundantly expressed in malignant tissues when compared to levels

of expression in normal tissues. At the protein level, greater immunoreactivity for

ghrelin and exon 3-deleted preproghrelin was found in human prostatectomy tumour

specimens than normal prostate tissue by immunohistochemistry (Chapter 3). This

was also found to be the case in breast cancer specimens when compared to normal

breast tissue (Chapter 4) and this suggests that ghrelin and exon 3-deleted

preproghrelin may represent novel diagnostic or prognostic immunohistochemical

markers for prostate and breast cancer.

Real-time RT-PCR is an effective, sensitive technique for the detection and

quantitation of low levels of mRNA expression (Pfaffl et al. 2002). Ghrelin mRNA is

known to be a low abundance mRNA species, therefore, we utilised this technique to

quantitate full-length preproghrelin and exon 3-deleted preproghrelin transcripts in

breast cancer cell lines of varying oncogenic phenotypes. Two methods of

quantitation are possible: absolute and relative quantitation. In this project, the

relative quantitation strategy was employed. This method is based on the expression

ratio of a target transcript versus a reference transcript (in this case 18S ribosomal

167

RNA). This technique is effective for detecting changes in the mRNA levels between

different samples and has been used successfully to investigate potential prognostic

markers in breast cancer (Peirce and Chen, 2001). Importantly, real time RT-PCR

demonstrated that exon 3-deleted preproghrelin transcript expression is significantly

up-regulated in highly malignant breast cancer cell lines (MDA-MB-231 and MDA-

MB-435) when compared to a benign breast epithelial cell line (MCF-10A). This

reflects our data generated from immunohistochemical studies on breast tissues and

provides more evidence for a role for the unique isoform in breast cancer, including

its potential as a novel diagnostic marker (Chapter 4).

Current mathematical models used to analyse data generated with the relative

quantitation strategy allow for only one transcript to be quantitated at a time – that is,

relative expression levels of a single transcript can be compared between samples,

however, multiple transcripts cannot. Software that can compare the relative

expression levels of up to four target genes in a sample has been developed to

circumvent this problem (Pfaffl et al. 2002) and could be useful for future studies.

This would allow the direct comparison of full-length and exon 3-deleted

preproghrelin transcript levels in one tissue and would allow us to determine if

transcription of the exon 3-deleted preproghrelin isoform is favoured over full-length

preproghrelin, as is the case for many alternatively-spliced transcripts in tumour

tissues (Venables, 2004). During the course of this project, real time RT-PCR

analysis of prostate cancer specimens for ghrelin and exon 3-deleted preproghrelin

expression was commenced; however, difficulty in obtaining prostate tumour

samples did not allow completion of this task. Full evaluation and validation of

ghrelin and exon 3-deleted preproghrelin as novel prognostic markers for prostate

and breast cancer requires further experiments with a larger number of

histopathological specimens. This work is clearly warranted given the data presented

in this thesis.

Identification of the ghrelin axis as a potential target for therapeutics in prostate and

breast cancer

The mechanism behind the proliferative effect of ghrelin in the LNCaP and

PC3 prostate cancer cell lines (Jeffery et al. 2002) was further explored in this study.

168

Ghrelin is the endogenous ligand for the GHS-R; a G protein-coupled receptor

(GPCR) (Kojima et al. 1999, Sun et al. 2004) that is expressed in prostate cancer cell

lines and tissues (Jeffery et al. 2002, Chapter 3) and may be involved in an

autocrine/paracrine loop in ghrelin-mediated prostate cancer cell proliferation

(Jeffery et al. 2002). GPCR signalling by various ligands can generate a mitogenic

response in prostate cell lines, often via crosstalk with mitogen activated protein

kinase (MAPK) pathways (Daaka, 2004). Phosphorylated (activated) MAPKs,

specifically the extracellular-signal regulated kinases (ERK 1 and 2), have been

implicated in prostate tumourigenesis, and growth factor-induced activation of

MAPK pathways results in increased cell proliferation and metastasis (Maroni et al.

2004). Murata and co-workers reported that ghrelin upregulates MAPK activity and

cell proliferation in the hepatoma cell line HepG2 (Murata et al. 2002) and therefore,

we speculated that this might also occur in prostate cancer cell lines (Figure 2). In

studies carried out primarily by an Honours student in our laboratory (Anthony Yeh),

it was shown that ghrelin did indeed rapidly activate the extracellular-signal

regulated kinases (ERKs) in the LNCaP and PC3 prostate cancer cell lines, an effect

that was completely abolished with specific MAPK inhibitors (Chapter 3, Yeh et al,

in press). The alternative MAPK pathway, the p38 kinase pathway, was also

activated (phosphorylated) in the LNCaP cell line after ghrelin treatment (Chapter 3).

Phosphorylated p38 MAPK activates transcription factors and cell-cycle regulators

which in turn influence cell proliferation, apoptosis and splicing events (Olson and

Hallahan, 2004). In the MCF-10A breast epithelial cell line, the p38 MAPK pathway

also interacts with the ERK 1/2 cascade resulting in increased cell migration and

invasion (Kim et al. 2003). Phospho-p38 MAPK can also activate the pro-survival

PI3K/AKT pathway (Horowitz et al. 2004) and ghrelin has also been shown to signal

through the PI3/AKT pathway in some cell types (Duxbury et al. 2003); therefore,

further work focusing on the downstream effects ghrelin-induced p38 activation in

prostate cancer is required.

Although ghrelin did not appear to protect prostate cancer cells from

chemically-induced apoptosis, the potential effect of ghrelin on apoptosis and cell

survival could be more accurately quantified using a Cell Death ELISAPLUS (Roche)

to measure DNA fragmentation and a Caspase 3 Activity Assay (Roche), both using

a fluorescence microplate reader (PolarSTAR, BMG). Direct observation of DNA

169

fragmentation could also be achieved by fluorescent microscopy using a TUNEL

assay (In situ cell death detection kit, Roche) on cells grown on coverslips. The

influence of ghrelin treatment on the regulation of genes that are anti-apoptotic (Bcl-

2/BclXL, NF-κB, IGF-I, caveolin and Akt) or pro-apoptotic (PTEN, p53, Bin1,

TGF-beta, and Par-4) in prostate cancer (Gurumurthy et al. 2001) could be

determined using real-time PCR, although not all of these genes are present in all of

the cell lines used in this study.

A number of studies have since been published demonstrating that ghrelin

augments cell proliferation via MAPK activation in a range of cell lines (Andreis et

al. 2003, Kim et al. 2004, Mazzocchi et al. 2004, Nanzer et al. 2004, Zhang et al.

2004). The finding that the proliferative effect of ghrelin in prostate cancer cells is

primarily a result of ghrelin-induced MAPK pathways is extremely significant given

that new treatments for hormone-refractory prostate cancer are urgently required,

with MAPK cascades being a promising new target for anti-cancer therapy (Maroni

et al. 2004) (Figure 2).

171

Ghrelin gene

Autocrine ghrelin

Paracrine ghrelin

Increased cell proliferation

Exon 3-deleted preproghrelin

Figure 2. A proposed model of ghrelin-mediated cell proliferation in prostate cancer

cells.

Ghrelin is produced by prostate cancer cells and is secreted into culture medium.

Secreted ghrelin may then act in an autocrine/paracrine fashion to stimulate MAPK

cascades via the GHS-R, which is also expressed in prostate cancer. Phosphorylation

of ERK 1 and 2 by exogenous ghrelin treatment stimulates prostate cancer cell

proliferation. Activated ERK 1 and 2 may also bind transcription factors (TF)

including ELK-1. Binding sites for ELK-1 exist on the ghrelin promoter (Kanamoto et

al. 2004); hence, autocrine/paracrine ghrelin action may also augment ghrelin

production, further driving cell proliferation. The novel exon 3-deleted preproghrelin

isoform is also expressed by prostate cancer cells, although its precise function is as

yet unknown. MAPK inhibitors decrease cancer cell proliferation (blue arrows) and

blocking of ghrelin production, secretion or signalling (red arrows) may also

antagonize MAPK cascades, thereby representing a novel therapeutic approach for

cancer in the future. This model may also be applicable to breast cancer cells and

other hormone-dependent cancer cell types.

GHS-RERK /MAPK

P

preproghrelin

inter-cascade cross-talk

TF

Inhibition of cell proliferation ?

MAPK inhibition

173

Ghrelin stimulates cultured pituitary cells to secrete growth hormone (GH)

(Kojima et al. 1999). GH is a potent mitogen for prostate cancer cells in vitro

(Untergasser et al. 1999), and has been identified as being an important oncogene in

breast cancer (Zhu et al. 2005). As prostate cancer cells have been shown to express

the GH axis (Chopin et al. 2002a), a commercial GH ELISA (Roche) was used to

determine if the addition of ghrelin to prostate cancer cells induced the secretion of

prostatic GH in vitro. Neither ghrelin nor C-terminal 3 peptide induced measurable

GH release from LNCaP or PC3 prostate cancer cell lines (data not shown).

However, investigating the effect of ghrelin treatment on expression of GH, and its

downstream mediator insulin-like growth factor I (IGF-I), in the prostate in vivo is an

important area of future study.

Functional assays demonstrating that ghrelin promotes proliferation in breast

cancer cell lines also supports previous work performed in prostate cancer cell lines

(Jeffery et al. 2002, Chapter 3). However, this contrasts with reports published by

other groups. In particular, ghrelin has been shown to inhibit cell proliferation of

breast and thyroid carcinoma cell lines in vitro (Cassoni et al. 2001, Volante et al.

2003). The proliferative actions of ghrelin appear to be cell-specific, possibly due to

GHS-R subtype expression and crosstalk between signalling pathways (Duxbury et

al. 2003, Maroni et al. 2004). The acylation status of ghrelin also clearly influences

the function of the peptide (Ariyasu et al. 2004) and the octanoic acid modification

of ghrelin is lost rapidly with non-optimum storage conditions (Hosoda et al. 2004).

Des-acyl ghrelin has recently been shown to have a negative impact on tissue growth

in an over-expressing mouse model, potentially via the modulation of octanoylated

ghrelin-induced GH secretion (Ariyasu et al. 2005). Evidence to support a clear role

for ghrelin in the mitogenesis of a range of cell lines in vitro now exists (see

Literature update, Chapter 2). The moderate yet significant increase in cell growth

post-ghrelin treatment may be due to the fact that autocrine production of

endogenous ghrelin (Figure 2) is sufficient to stimulate proliferation, as is postulated

to be the case with the human erythroleukemic cell line HEL (DeVriese et al. 2005).

The use of anti-sense technology to downregulate ghrelin expression could help to

clarify this issue in the future. Indeed, inhibition of ghrelin signalling may have

therapeutic potential in prostate and breast (Chopin et al. 2002b); however, effective

ghrelin/GHS-R antagonists were not available during the course of this project.

174

Potent and selective ghrelin analogues that bind to the GHS-R and inhibit ghrelin

signalling have recently been developed (Halem et al. 2004, Liu et al. 2004) and may

prove useful for inhibition of the ghrelin axis in prostate and breast cancer.

Treatment of prostate and breast cancer cell lines with the C-terminal peptide

of exon 3-deleted preproghrelin (C-terminal 3 peptide) had no effect on

proliferation or MAPK phosphorylation, and the primary role of the isoform may

only be to produce the mature ghrelin peptide (Figure 1). I have established a

continuous cell line derived from normal epithelial prostate cells (RWPE-1, Bello et

al. 1997) and the PC3 prostate cancer cell lines, transfected with exon 3-deleted

preproghrelin mRNA using a tetracycline-regulated expression system for

mammalian cells (T-Rex, Invitrogen). The phenotype of these cell lines is currently

being investigated and will allow a more thorough functional analysis of the novel

isoform in the future. Prostate cancer cell lines overexpressing full-length

preproghrelin have also been generated in our laboratory, although due to time

constraints, their phenotypes have yet to be examined. Migration of RWPE-1 cells

towards a chemoattractant (bovine calf serum) is increased in the presence of

exogenous ghrelin (data not shown) and therefore it would be interesting to examine

the migratory properties of these overexpressing cell lines and the behaviour of

breast cancer cell lines transfected with these constructs. Future studies involving the

subcutaneous or subcapsular renal injection of the transfected cells into nude or

SCID mice would also be valuable in assessing the metastatic potential of prostate or

breast cancer cells overexpressing ghrelin and or exon 3-deleted preproghrelin in an

in vivo model.

This project has relied heavily on the use of experimentally immortalized cell

lines for in vitro functional assays and for extraction of RNA and protein. It is a

regular criticism that cell-based assays are not physiologically relevant and that cell

line models are not valid representations of disease in vivo. The immortalization of

cells, essential to circumvent natural cell senescence, often results in karyotypic

change, a transformed phenotype, and selection of non-representative cell

populations (Navone et al. 1999, Gudjonsson et al. 2004). However, transformed cell

lines are relatively inexpensive to maintain, safe to manipulate and provide valuable

preliminary in vitro data, often providing the justification for more expensive and

175

difficult in vivo investigations. Although the availability of human tumour tissue

samples has been limited over the course of this project, data generated from cell

lines and in vitro cell assays have allowed us to establish excellent collaborations

with the medical fraternity, thereby providing substantial resources of clinical

samples for future work. In addition, the future use of primary cell cultures,

multicellular spheroids (three-dimensional aggregates of cells) and anchorage-

independent cell culture (via soft agar assays) may also provide a better model of in

vivo tumour behaviour than traditional monolayer cell culture. For example, spheroid

cell culture has been shown to more accurately reflect tumour microenvironment and

stromal-epithelial interactions (Hamilton, 1998, Bates et al. 2000). A more accurate

depiction of the role of the ghrelin axis in hormone-dependent cancer is likely to be

generated, however, through the future use of in vivo mouse models.

The characterisation of the ghrelin axis in the mouse for the future development of

mouse models of cancer.

Mouse models of prostate and breast cancer are important tools for studying

the mechanisms behind early and late carcinogenesis (Abate-Shen and Shen, 2002,

Cardiff et al. 2003), particularly when there are difficulties in accessing human tissue

specimens. They also allow in vivo investigations into the physiological effects of

administration of compounds, as well as chemoprevention studies, which would be

otherwise unfeasible. In the TRAMP (Transgenic Adenocarcinoma Mouse Prostate)

mouse model, specific expression of the oncogene SV40 T antigen in prostate

epithelia is driven by the prostate-specific probasin promoter and results in the

development of prostate cancer and subsequent distant site metastases (Greenberg et

al. 1995). Interrogation of signalling pathways in TRAMP mice has demonstrated

the importance of growth factors including IGF-I in prostate cancer progression

(Foster et al. 1999) and it would be interesting to investigate in vivo ghrelin/GHS-R

expression and signalling in this manner. To ensure that future studies of the ghrelin

axis in this and other mouse models of cancer are warranted, we examined the

expression of the axis in a wide range of mouse tissues (Chapter 5, Jeffery et al.

2005). This report is the first to fully characterise the expression of ghrelin, a murine

homologue of exon 3-deleted preproghrelin, and the GHS-R type 1a in the mouse.

This will serve as an important reference for the examination of the role of the

176

ghrelin axis in development/progression of prostate cancer and other hormone-

dependent cancers, including breast cancer. This work has also generated important

collaborations with international research groups. Ongoing work includes in vivo

mouse studies to investigate the potential function of the exon 3-deleted

preproghrelin variant via injection of the novel isoform into mice and binding assays

investigating its affinity for orphan G protein-coupled receptors.

In summary, this study has provided new and compelling evidence that

supports a role for the ghrelin axis in hormone-dependent cancer and specifically in

prostate and breast cancer. We have identified several potential diagnostic markers

for prostate and breast cancer within the ghrelin axis. Ghrelin stimulates proliferation

of hormone-dependent cancer cells and therefore the ghrelin axis represents a novel

target for the treatment of such cancers (Figure 2). This project has also provided a

basis for future in vivo work in mouse models, which will potentially aid in the

development of new adjunctive therapies for prostate and breast cancer.

177

REFERENCES

Abate-Shen C, Shen MM (2002) Mouse models of prostate carcinogenesis. Trends in

Genetics 18: 1-5.

Andreis PG, Malendowicz LK, Trejter M, Neri G, Spinazzi R, Rossi GP, Nussdorfer

GG (2003) Ghrelin and growth hormone secretagogue receptor are expressed in the

rat adrenal cortex: Evidence that ghrelin stimulates the growth, but not secretory

activity of adrenal cells. FEBS Letters 536: 173-179.

Ariyasu H, Takaya K, Iwakura H, Hosoda H, Akamizu T, Arai Y, Kangawa K &

Nakao K (2005) Transgenic mice overexpressing des-acyl ghrelin show small

phenotype. Endocrinology 146: 355-364.

Bates RC, Edwards NS, Yates JD (2000) Spheroids and cell survival. Critical

Reviews in Oncology/Hematology 36: 61-74.

Bello D, Webber MM, Kleinman HK, Wartinger DD, Rhim JS (1997). Androgen

responsive adult human prostatic epithelial cell lines immortalized by human

papillomavirus 18. Carcinogenesis 18: 1215-1223.

Cardiff RD (2003) Mouse models of human breast cancer. Comparative Medicine

53: 250-253.

Cassoni P, Papotti M, Ghe C, Catapano F, Sapino A, Graziani A, Deghenghi R,

Reissmann T, Ghigo E, Muccioli G (2001). Identification, characterisation and

biological activity of specific receptors for natural (ghrelin) and synthetic growth

hormone secretagogues and analogs in human breast carcinoma and cell lines.


Chopin LK, Veveris-Lowe TL, Philipps AF, Herington AC (2002a) Co-expression of

GH and GHR isoforms in prostate cancer cell lines. Growth Hormone and IGF

Research 12: 126-136.

178

Chopin LK, Jeffery PL, Herington AC (2002b) Reproductive cancer diagnosis and

therapy. International Patent PCT/AU02/00582 (14/11/2002).

Coradini D, Daidone MG (2004) Biomolecular prognostic factors in breast cancer.

Current Opinions in Obstetrics and Gynecology. 16: 49-55.

Daaka Y (2004) G proteins in cancer: the prostate cancer paradigm. Science “Signal

Transduction Knowledge Environment” 216: 2.

De Vriese C, Gregoire F, DE Neef P, Robberecht P, Delporte C (2005) Ghrelin is

produced by the human erythroleukemic HEL cell line and involved in an autocrine

pathway leading to cell proliferation. Endocrinology 146: 1514-1522.

Docherty K and Steiner DF (1982) Post-translational proteolysis in polypeptide

hormone biosynthesis. Annual Review of Physiology 44: 625-638.

Duxbury MS, Waseem T, Ito H, Robinson MK, Zinner MJ, Ashley SW, Whang EE

(2003) Ghrelin promotes pancreatic adenocarcinoma cellular proliferation and

invasiveness. Biochemical and Biophysical Research Communications 309: 464-468.

Epstein JL (2004) Diagnosis and reporting of limited adenocarcinoma of the prostate

on needle biopsy. Modern pathology: an official journal of the United States and

Canadian Academy of Pathology, Inc: 307-315.

Foster BA, Kaplan PJ, Greenberg NM (1999) Peptide growth factors and prostate

cancer: new models, new opportunities. Cancer and Metastasis Reviews 17: 317-324.

Greenberg NM, De Mayo F, Finegold MJ, Medina D, Tilley WD, Aspinall JO,

Cunha GR, Donjacour AA, Matusik RJ, Rosen JM (1995) Prostate cancer in a

transgenic mouse. Proceedings of the National Academy of Sciences of the United

States of America 92: 3439-3443.

179

Gudjonsson T, Villadsen R, Ronnov-Jessen L, Petersen OW (2004) Immortalization

protocols used in cell culture models of human breast morphogenesis. Cellular and

Molecular Life Sciences 61: 2523-2534.

Gurumurthy S, Vasudevan KM, Rangnekar VM (2001) Regulation of apoptosis in

prostate cancer. Cancer Metastasis Reviews 20:225-43.

Halem HA, Taylor JE, Dong JZ, Shen Y, Datta R, Abizaid A, Diano S, Horvath T,

Zizzari P, Bluet-Pajot M, Epelbaum J, Culler MD (2004) Novel analogs of ghrelin:

physiological and clinical implications. European Journal of Endocrinology 151:71-

75.

Hamilton G (1998) Multicellular spheroids as an in vitro tumor model. Cancer letters

131: 29-34.

Horowitz JC, Lee DY, Waghray M, Keshamouni VG, Thomas PE, Zhang H, Cui Z,

Thannickal VJ (2004) Activation of the pro-survival phosphatidylinositol 3-

kinase/AKT pathway by transforming growth factor-beta 1 in mesenchymal cells is

mediated by p38 MAPK-dependent induction of an autocrine growth factor. Journal

of Biological Chemistry 279: 1359-1367.

Hosoda H, Doi K, Nagaya N, Okumura H, Nakagawa E, Enomoto M, Ono F,

Kangawa K (2004) Optimum collection and storage conditions for ghrelin

measurements: octanoyl modification of ghrelin is rapidly hydrolyzed to desacyl

ghrelin in blood samples. Clinical Chemistry 50: 1077-1080.


hormone-releasing peptide ghrelin and the growth hormone secretagogue receptor in

prostate cancer cell lines. Journal of Endocrinology 172: 7-11.



deleted proghrelin variant encondes a novel C-terminal peptide. Endocrinology 146:

432-440.

180

Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, Feuer EJ, Thun MJ

(2005) Cancer Statistics, 2005. CA: A cancer journal for clinicians 55: 10-30.

Kanamoto N, Akamizu T, Tagami T, Hataya Y, Moriyama K, Takaya K, Hosoda H,

Kojima M, Kangawa K, Nakao K (2004) Genomic structure and characterization of

the 5'-flanking region of the human ghrelin gene. Endocrinology 145: 4144-4153.

Kim MS, Lee EJ, Kim HR, Moon A (2003) p38 kinase is a key signaling molecule

for H-Ras-induced cell motility and invasive phenotype in human breast epithelial

cells. Cancer Research 63: 5454-5461.

Kim MS, Yoon CY, Jang PG, Park YJ, Shin CS, Park HS, Ryu JW, Pak YK, Park JY

Lee KU, Kim SY, Lee HK, Kim YB, Park KS (2004) The mitogenic and

antiapoptotic actions of ghrelin in 3T3-L1 adipocytes. Molecular Endocrinology 18:

2291-2301.

Kojima M, Hosodo H, Date Y, Nakazato M, Matsuo H, Kanagawa, K (1999) Ghrelin

is a growth hormone releasing acylated peptide from stomach. Nature 402: 656-660.

Liu B, Liu G, Xin Z, Serby MD, Zhao H, Schaefer VG, Falls HD, Kaszubska W,

Collins CA, Sham HL (2004) Novel isoxazole carboxamides as growth hormone

secretagogue receptor (GHS-R) antagonists. Bioorganic and Medicinal Chemistry

Letters 14: 5223-5226.

Maroni PD, Koul S, Meacham RB, Koul H (2004) Mitogen activated protein kinase

signal transduction pathways in the prostate. Cell Communication and Signaling 2:5-

18.

Mazzocchi G, Neri G, Rucinski M, Rebuffat P, Spinazzi R, Malendowicz LK,

Nussdorfer GG (2004) Ghrelin enhances the growth of cultured human adrenal zona

glomerulosa cells by exerting MAPK-mediated proliferogenic and antiapoptotic

effects. Peptides 25: 1269-1277.

181

Murata M, Okimura Y, Iida K, Matsumoto M, Sowa H, Kaji H, Kojima M, Kangawa

K, Chihara K (2002) Ghrelin modulates the downstream molecules of insulin

signaling in hepatoma cells. Journal of Biological Chemistry 277: 5667-5674.

Nanzer AM, Khalaf S, Mozid AM, Fowkes RC, Patel MV, Burrin JM, Grossman A,

Korbonits M (2004) Ghrelin exerts a proliferative effect on a rat pituitary

somatotroph cell line via the mitogen-activated protein kinase pathway. European


Navone NM, Logothetis CJ, von Eschenbach AC, Troncoso P (1999) Model systems

of prostate cancer: Uses and limitations. Cancer and Metastasis Reviews 17: 361-

371.

Olson JM, Hallahan AR (2004) p38 MAP kinase: a convergence point in cancer

therapy. Trends in Molecular Medicine 10: 125-129.

Pemberton C, Wimalasena P, Yandle T, Soule S, Richards M (2003) C-terminal pro-

ghrelin peptides are present in the human circulation. Biochemical and Biophysical

Research Communications. 310: 567-573.

Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool

(REST©) for group-wise comparison and statistical analysis of relative expression

results in real-time PCR. Nucleic Acids Research 30: 36-46.

Pierce SK, Chen WY (2001) Quantification of prolactin receptor mRNA in multiple

human tissues and cancer cell lines by real time RT-PCR. Journal of Endocrinology

171: 1-4.

Seidah NG, Chretien M (1997) Eukaryotic protein processing: endoproteolysis of

precursor proteins. Current Opinions in Biotechnology 8: 602-607.

Sivridis E, Touloupidis S, Giatromanolaki A (2002) Immunopathological prognostic

and predictive factors in prostate cancer. International Urology and Nephrology 34:

63-71.

182

Sun Y, Wang P, Zheng H, Smith RG (2004) Ghrelin stimulation of growth hormone

release and appetite is mediated through the growth hormone secretagogue receptor.

Proceedings of the National Academy of Sciences of the United States of America

101: 4679-4684.

Tanaka M, Hayashida Y, Iguchi T, Nakao N, Nakai N, Nakashima K (2001)

Organization of the mouse ghrelin gene and promoter: occurrence of a short

noncoding first exon. Endocrinology 142: 3697-3700.

Thompson IM, Pauler DK, Goodman PJ, Tangen CM, Lucia MS, Parnes HL,

Minasian LM, Ford LG, Lippman SM, Crawford ED, Crowley JJ, Coltman CA

(2004) New England Journal of Medicine 350: 2239-2246.

Untergasser G, Rumpold H, Hermann M, Dirnhofer S, Jilg G, Berger P (1999)

Proliferative disorders of the aging human prostate: involvement of protein hormones

and their receptors. Experimental Gerontology 34: 275-287.

Venables JP (2004) Aberrant and alternative splicing in cancer. Cancer Research 64:

7647-7654.

Volante M, Allia E, Fulcheri E, Cassoni P, Ghigo E, Muccioli G, Papotti M (2003)

Ghrelin in fetal thyroid and follicular tumors and cell lines: expression and effects on

tumor growth. American Journal of Pathology 162: 645-654.

Watson RWG, Schalken JA (2004) Future opportunities for the diagnosis and

treatment of prostate cancer. Prostate Cancer and Prostatic Diseases. 7 (Suppl 1): 8-

13.

Yeh AH, Jeffery PL, Duncan RP, Herington AC, Chopin LK (2005) Ghrelin and a

novel preproghrelin isoform are highly expressed in prostate cancer compared to

normal prostate tissue and ghrelin-mediated prostate cancer cell line proliferation

involves the MAPK pathway. Clinical Cancer Research 2005 in press

183

Zhang W, Lin TR, Hu Y, Fan Y, Zhao L, Stuenkel EL, Mulholland MW (2004)

Ghrelin stimulates neurogenesis in the dorsal motor nucleus of the vagus. Journal of

Physiology 559: 729-737.

Zhu T, Starling-Emerald B, Zhang X, Lee KO, Gluckman PD, Mertani HC, Lobie PE

(2005) Oncogenic transformation of human mammary epithelial cells by autocrine

human growth hormone. Cancer Research 65: 317-324.

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