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Biochimica et Biophysica Acta 1704 (2004) 1–10

Review

Gastrins, cholecystokinins and gastrointestinal cancer

Ahmad Aly, Arthur Shulkes, Graham S. Baldwin*

Department of Surgery, University of Melbourne, Austin Campus, A&RMC, Studley Road, Heidelberg, Melbourne, Victoria 3084, Australia

Received 26 May 2003; received in revised form 15 January 2004; accepted 21 January 2004

Available online 5 February 2004

Abstract

The gastrointestinal peptide hormones gastrin and cholecystokinin (CCK) are well known for their ability to stimulate gastric acid

secretion and pancreatic enzyme secretion, respectively. The suggestion that gastrin and CCK might also promote the development of cancers

of the gastrointestinal tract has been controversial, but an increasing body of evidence now supports the view that the amidated and non-

amidated forms of gastrin act as growth factors via different receptors in different regions of the gut. For example, animal experiments

indicate that amidated gastrins are involved in cellular differentiation and repair in the gastric mucosa, and synergise with Helicobacter pylori

infection in the development of gastric carcinoma. In contrast, non-amidated gastrins stimulate colonic mucosal growth, accelerate the early

steps in colorectal carcinoma formation, and are elevated in the tumour and circulation of patients with colorectal cancer. Although human

pancreatic carcinomas express CCK-1 and CCK-2 receptors, the role of gastrins and CCK in pancreatic carcinogenesis is yet to be

established. Further investigation of the possible role of the CCK-2 receptor in gastric and pancreatic neoplasia, and of the hypothesis that

gastrin precursors act as autocrine growth factors in colorectal carcinoma, is warranted. However, therapies aimed at the gastrins must be

targeted to the relevant gastrin/gastrin receptor combination.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Colorectal cancer; Gastric cancer; Gastrin; Migration; Pancreatic cancer; Proliferation

1. Introduction

Gastrin is a classical gut peptide hormone, which was

identified originally as a stimulant of gastric acid secretion. It

is produced principally by the G cells of the gastric antrum

[1], and to a variable extent in the upper small intestine, with

much lower amounts in the colon and pancreas. The related

hormone cholecystokinin (CCK), which has the same C-

terminal tetrapeptide amide as gastrin, is synthesised in the

duodenum and is responsible for pancreatic enzyme secre-

tion. Amidation is essential for the stimulatory effects of

gastrin on gastric acidity and CCK on pancreatic secretion.

Over the last two decades, the realisation that gastrin

played a crucial role in the development of gastric carcinoids

generated much interest in the possibility that gastrin might

also influence the development and growth of gastric cancers

[2]. The observation of increased serum gastrin concentra-

tions in patients with colorectal cancer suggested that gastrin

0304-419X/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbcan.2004.01.004

Abbreviations: CCK, cholecystokinin; COX-2, cyclooxygenase-2;

Gamide, amidated gastrin17; Ggly, glycine-extended gastrin17* Corresponding author. Tel.: +613-9496-5592; fax: +613-9458-1650.

E-mail address: grahamsb@unimelb.edu.au (G.S. Baldwin).

might also be a key peptide in colorectal carcinogenesis. The

evidence, however, was not clear and the debate surrounding

the role of gastrin in gastrointestinal neoplasia was enthusi-

astically argued but remained unresolved.

Recent findings have added a new dimension to the

debate. The observation that gastrin precursors such as

progastrin and glycine-extended gastrin (Ggly) may mediate

a growth effect in colonic epithelium has provided the

impetus to reexamine the evidence for the potential role of

‘‘gastrins’’ in gastrointestinal cancer. The possibility that

past conflicting findings may be reconciled by studying

gastrin precursors as well as gastrin is considered in the

present review, which emphasises the 5 years since we last

reviewed this field [2].

2. Structure and biosynthesis of gastrin peptides

Like many other peptide hormones, the initial translation

product of the gastrin gene is a large precursor molecule,

preprogastrin (101 amino acids), which is converted to

progastrin (80 amino acids) by cleavage of the N-terminal

signal peptide (Fig. 1). Progastrin is processed further within

Fig. 1. Gastrin processing. Preprogastrin (101 amino acids) is converted to progastrin (80 amino acids) by removal of the signal peptide. The sequential action

of endopeptidases and carboxypeptidase B-like enzymes then converts progastrin to glycine-extended forms. The extent of conversion of glycine-extended to

amidated forms is dependent on the tissue. Both non-amidated and amidated forms are independently active via different receptors.

A. Aly et al. / Biochimica et Biophysica Acta 1704 (2004) 1–102

secretory vesicles by endo- and carboxy-peptidases to yield

glycine-extended gastrins. The C-terminus of glycine-ex-

tended gastrin34 is then amidated by peptidyl a-amidating

monooxygenase, and further proteolytic cleavage results in

mature amidated gastrin17 (Gamide). In healthy humans,

progastrin and the glycine-extended gastrins comprise less

than 10% of circulating gastrins.

Progastrin and its glycine-extended derivatives have

previously been regarded as physiologically inactive. How-

ever, data have been accumulating to suggest that gastrin

precursors such as Ggly stimulate proliferation in several

cancer cell lines [3,4]. Interestingly, Ggly binds two ferric

ions with high affinity [5], and ferric ion binding is essential

for biological activity [6]. The observation that CCK-2

receptor antagonists did not inhibit the growth response to

Ggly suggests that novel receptors, distinct from classical

gastrin receptors, are involved. The preparation of recom-

binant human progastrin by two independent groups [7,8]

should assist in the definition of the relationship between

Ggly and progastrin receptors.

3. Structure and location of gastrin receptors

Amidated gastrins bind with high affinity to CCK-1 and

CCK-2 receptors (previously referred to as CCK-A and

CCK-B receptors, respectively), which can be differentiated

by the substantially greater affinity of the CCK-1 receptor

for CCK (see Ref. [9] for review). Both receptors belong to

the family of seven transmembrane domain receptors, and

share 50% identity in sequence. In addition, both amidated

and non-amidated gastrins [10] bind to a low affinity

receptor, often referred to as the CCK-C receptor, which is

present in a variety of tissues, including neoplastic cell lines

[11]. The CCK-C receptor, which is a likely target for the

inhibitory effects of the nonselective gastrin antagonists

proglumide and benzotript on the growth of colon carcino-

ma cells in vitro [12], is unrelated in structure to the

classical gastrin receptors, and belongs to the family of

enzymes involved in h-oxidation of fatty acids [13].

Initial confusion over the type and number of CCK

receptors expressed in the gastrointestinal tract has been

clarified over the last 5 years. In the fundus of the stomach,

CCK-2 receptors have been identified on parietal, entero-

chromaffin-like (ECL) and D cells, while in the antrum they

are confined to D cells [14–17]. CCK-1 receptors are

present on fundic chief cells and antral D cells. Although

CCK-1 receptor mRNA has also been detected in human

pancreas by PCR by some workers [18], but not by other

groups [19], pancreatic acini do not express significant

numbers of functional CCK-1 receptors [20]. The normal

human pancreas does contain both CCK-2 receptor mRNA

[19] and CCK-2 receptors [20], which are localised to the

glucagon-producing cells of the pancreatic islets [21]. The

normal colon does not seem to express gastrin receptors

frequently, since CCK-2 receptor mRNA was detected by

PCR in only 2/15 samples (13%) [22].

4. Role of gastrins in growth and repair of normal

mucosa

Gamide is an important trophic factor for gastric epithe-

lium, and is known to stimulate proliferation of the ECL

cells of the stomach and proximal small intestine [23], and

migration of gastric parietal cells [24]. The proliferative

effect can result in carcinoid tumour formation secondary to

prolonged hypergastrinaemia in conditions such as Zollin-

ger–Ellison syndrome and pernicious anaemia [25]. In

contrast, Gamide does not appear to have a significant

proliferative role in other regions of the gastrointestinal

tract. Both exogenously administered gastrin in vivo and

A. Aly et al. / Biochimica et Biophysica Acta 1704 (2004) 1–10 3

omeprazole-induced hypergastrinaemia cause gastric hyper-

plasia without significant increases in intestinal, pancreatic

or colonic size in several species [26,27]. The increase in

pancreatic weight observed after fundectomy does not

appear to be caused by the resultant hypergastrinaemia, as

a similar increase in pancreatic weight was seen after total

gastrectomy, which results in hypogastrinaemia [28]. How-

ever, Gamide does appear to act as a mitogen for the

metaplastic ductular cells generated in vivo by ligation of

rat pancreatic ducts [29,30].

The role of CCK in pancreatic development needs to be

clarified. In homozygous CCK-deficient mice, pancreatic

weight and cell morphology appeared normal [31]. Simi-

larly, in CCK-1 receptor-deficient mice both acinar gland

and islet morphologies were comparable to the normal

pancreas [32]. However, Otsuka Long–Evans Tokushima

(OLETF) rats, in which a naturally occurring deletion has

disrupted the CCK-1 receptor gene [33], develop pancreatic

ductal hyperplasia. Genetic analysis suggests an association

between the hyperplasia and the presence of a disrupted

CCK-1 receptor gene [34].

Growth effects of non-amidated gastrins have also been

demonstrated in colonic tissue, both in vivo and in vitro.

Homozygous gastrin-deficient mice have histologically nor-

mal colonic mucosa but reduced proliferative index as

measured by BrdU labelling [35]. This observation suggests

that the gastrin gene influences proliferation of the colonic

mucosa but does not define the form of gastrin involved.

Although infusion of Gamide into gastrin-deficient mice

had no effect on the colonic proliferative index, infusion of

Ggly increased the proliferative index by 80%. Transgenic

mice overexpressing human progastrin in the liver have

high concentrations of circulating progastrin but normal

Gamide concentrations. These mice have thickened colonic

mucosa with deeper crypts and an increased proliferative

index in both proximal and distal colon compared to wild-

type mice [36]. Similar results have been reported for

transgenic mice overexpressing Ggly [37]. Since these

transgenic mice have had high progastrin exposure since

infancy, it could be argued that the results reflect develop-

mental and/or lifelong exposure. Although our own studies

have confirmed the proliferative effect of infused Ggly upon

the colon of normal young adult rats even after 1-month

exposure [38], a recent report found that Ggly infusion for 7

days resulted in an increase in colonic weight, but had little

effect on proliferation [27].

5. Effect of gastrins on proliferation of gastrointestinal

carcinoma cell lines

Although recent studies with gastric carcinoma cell lines

have demonstrated the presence of gastrin mRNA, and

processed and unprocessed gastrin [39], growth of the

majority of lines is not stimulated by exogenous Gamide

(reviewed in Ref. [40]). Recently both Gamide and Ggly

were shown to stimulate growth of three human gastric cell

lines. The effects of Gamide on AGS [41] and IMGE [42]

cells were mediated by the CCK-2 receptor, while Gamide

stimulated SIIA cells via a CCK-1-like receptor [41]. The

observation that CCK-1 or CCK-2 receptor antagonists did

not block the proliferative effect of Ggly suggested the

involvement of a novel receptor [41,42].

The expression and role of gastrins and their receptors in

pancreatic carcinoma cell lines continue to be controversial

(see Ref. [40] for review of early work in this area).

Although CCK-2 receptor mRNA was detected by PCR in

8/8 [43] and 6/6 [44] human pancreatic carcinoma cell lines,

a more recent report found that only 2/14 lines were positive

[45]. A similar discrepancy exists for CCK-1 receptor

mRNA [43,45]. While some well-established human pan-

creatic carcinoma cell lines (e.g. PANC-1 [46,48], MIA

PaCa-2 [47,48], BxPC-3 [49,50]) clearly respond to gastrin

or CCK via a CCK-2 receptor-dependent pathway, great

heterogeneity was observed in the proliferative responses of

a newly developed panel of pancreatic cell lines to gastrin,

CCK, and selective CCK-1 and CCK-2 receptor antagonists

[51]. Moreover, the failure to detect gastrin immunoreactiv-

ity in conditioned media from a panel of 14 human

pancreatic carcinoma cell lines argues against the general

occurrence of an autocrine loop involving gastrin and the

CCK-2 receptor [45].

Most colorectal carcinoma cell lines neither express

CCK-2 receptors nor respond to Gamide (reviewed in Ref.

[40]). In contrast, Ggly stimulated growth of approximately

50% of the colorectal cell lines tested [52–54] via a receptor

distinct from the CCK-2 receptor [53–55].

6. Effect of gastrins on migration of cancer cell lines

The possibility that gastrins might modulate cell migra-

tion and invasiveness has only recently been investigated.

The first clue came from the observation that the CCK-1

receptor antagonist loxiglumide reduced both the expression

of matrix metalloproteinase-9 (MMP-9) and the invasive-

ness of two human pancreatic carcinoma cell lines [56] via a

protein kinase C-dependent pathway [57]. Similar results

have been obtained following Gamide treatment of a CCK-2

receptor-transfected subline of the human gastric cancer cell

line AGS [58]. Gamide stimulation of AGS-CCK-2 receptor

cells cultured on artificial basement membrane led to

branching morphogenesis [59]. The effects of Gamide on

cell migration may also contribute to the enhancement of

cryoulcer healing observed in rat gastric mucosa [60].

Interestingly, only non-amidated gastrins such as Ggly were

able to stimulate migration of the human colorectal carci-

noma cell line LoVo [61] and the mouse gastric cell line

IMGE-5 [42]. The pathway in the latter case involved

phosphorylation of the adherens junction protein h-catenin,which has been implicated in the effects of the mutated APC

gene product in colorectal cancer (see below).

A. Aly et al. / Biochimica et Biophysica Acta 1704 (2004) 1–104

7. Gastrin and gastric carcinoids

Gamide stimulates proliferation of gastric ECL cells, the

major histamine-producing endocrine cell of the oxyntic

mucosa, by interacting with the CCK-2 receptor [62].

Sustained hypergastrinaemia in the rat associated with

continuous administration of H2 receptor antagonists or

proton pump inhibitors results in ECL cell hyperplasia

within a few weeks and ECL cell carcinoids after 12 months

[63]. However, the human is less sensitive to these inhibitors

of gastric acid production, with 5 years of proton pump

treatment resulting in ECL cell hyperplasia rather than

carcinoids [63]. Chronic hypergastrinaemia in humans al-

ways results in some ECL cell hyperplasia. However, the

observation that the extent of ECL cell changes in the

progression hyperplasia–dysplasia–carcinoid is dependent

on the cause of the hypergastrinaemia suggests that factors

other than gastrin are involved. Thus, ECL cell carcinoids

are detected in about 5% of subjects with atrophic gastritis

associated with pernicious anaemia, but in 40% of patients

with Zollinger–Ellison syndrome as part of the MEN Type I

syndrome [63]. In contrast, patients with sporadic Zollin-

ger–Ellison syndrome rarely have carcinoids [64].

Table 1

Frequency of gastrin-, CCK- and CCK receptor-positive gastrointestinal carcinom

Percentage positive for

Gastrin mRNA Progastrin Ggly Gamide CCK

mRNA

CCK-1

recepto

Gastric carcinoma

– – – 36 – –

0 – – – 29 36

– – – – – 0

– 34 35 38 – –

Pancreatic carcinoma

– – – – – –

– – – – – 100

– – – – – 90

– 91 55 23 – –

– 100 63 74 0 67

Colorectal carcinoma

– – – – – –

– – – 21 – –

– – – 0 – –

– 100 100 100 – –

– 87 – 97 – –

– – 45 43 – –

– 100 0 8 – –

– – – – – –

– 100 44 69 – –

86 – – – – –

87 – – – – –

– – – – – 0

– – – – – 0

44 – – – – –

FC, flow cytometry; IHC, immunohistochemistry; – , not measured; PCR, polym

RIA, radioimmunoassay; RPA, RNase protection assay.

8. Role of gastrins in development and progression of

gastric cancer

Although a genetic model of the progression to gastric

cancer has not been established, evidence is accumulating

that gastric cancer (of the intestinal or well-differentiated

type in particular) is initiated by atrophic gastritis mediated

by Helicobacter pylori [65,66]. The risk of gastric cancer is

dependent on the strain of H. pylori, and is increased when

bacterial colonization is located predominantly in the cor-

pus. However, the observation that only a small percentage

of infected subjects develop gastric cancer indicates that

progression to gastric cancer is also modulated by environ-

mental factors and by the inflammatory response [67,68].

Significant studies over the last few years suggest that

gastrin is an important factor in determining the progression

to gastric cancer. One consequence of the decreased acid

output that follows H. pylori colonization of the corpus is an

unopposed increase in gastrin, which in turn directly stim-

ulates H. pylori proliferation [69]. Wang et al. [70] made the

important observation that chronic hypergastrinaemia in

transgenic mice overexpressing Gamide results in a pro-

gression from hyperchlorhydria to hypochlorhydria, gastric

as

Number Method Reference

r

CCK-2

receptor

CCK-C

receptor

of samples

– – 22 FC [72]

7 – 14 PCR [73]

7 – 27 RA [74]

34 – 15 IHC [75]

0 – 12 RA [76]

100 – 22 PCR [19]

– – 30 PCR [77]

95 – 15 IHC [78]

100 – 19 PCR,

RIA

[79]

57 – 67 RB [80]

– – 28 FC [72]

– – 16 RIA [81]

– – 15 RIA [82]

– – 23 IHC [83]

– – 44 RIA [84]

– – 12 RIA [85]

20 – 10 PCR [86]

– – 32 RIA [87]

11 75 112 RPA [88]

77 100 30 PCR [89]

100 – 6 PCR [19]

4 – 25 RA [74]

38 – 79 PCR [22]

erase chain reaction; RA, receptor autoradiography; RB, receptor binding;

A. Aly et al. / Biochimica et Biophysica Acta 1704 (2004) 1–10 5

atrophy and finally gastric cancer, and that the progression

is accelerated by concurrent Helicobacter infection. Hence,

elevated gastrin may be not only a consequence but also a

cause of gastric atrophy. The mechanism is unclear but may

be related to gastrin-mediated increases in the concentra-

tions of the growth factors heparin binding-epidermal

growth factor and transforming growth factor a. Interest-

ingly, a recent report from the same group indicates that

gastric cancer occurs in male mice only [71].

The presence of gastrins and their receptors in gastric

adenocarcinomas remains a controversial issue (Table 1).

Gamide, progastrin, Ggly and the CCK-2 receptor have

been detected in human gastric adenocarcinomas by immu-

nohistochemistry [75] with an increased proportion of

positive cells in the progression from intestinal metaplasia

to adenocarcinoma. However, CCK-2 receptor expression

was detected in only 7% of gastric cancer samples by RT-

PCR [73], or by receptor autoradiography [74].

9. Role of gastrins in development and progression of

pancreatic cancer

Most human pancreatic adenocarcinomas express both

gastrin mRNA and progastrin-derived peptides [78,79] (Ta-

ble 1). Concentrations of gastrin mRNAwere greater than in

normal pancreas, as detected by PCR. Immunohistochemis-

try with region-specific antisera revealed progastrin, Ggly

and Gamide in 91%, 55%, and 23% of tumours, respectively

[78]. Gamide was detected in 14/19 (74%) tumour extracts

with the median concentration being 0.4 pmol/gm [79].

However, no increase in serum Gamide concentrations

was detected in patients with pancreatic adenocarcinoma

[90]. Neither CCK precursors nor amidated CCK were

detected in extracts of normal pancreas or pancreatic ade-

nocarcinoma by radioimmunoassay [79], and there was no

significant difference in serum CCK between patients with

pancreatic adenocarcinoma at different stages [91].

There is now general agreement (Table 1) that the great

majority of pancreatic adenocarcinomas express both CCK-

1 and CCK-2 receptors [19,77–79]. In contrast, gastrinomas

generally do not express CCK-2 receptors, although CCK-1

receptors have been detected in 40% of specimens [92,93].

Other gastroenteropancreatic neuroendocrine tumours ex-

press CCK-1 receptors in 36%, and CCK-2 receptors in

32%, of specimens [93].

Many lines of evidence suggest that the CCK-1 receptor

may be involved in chemically induced pancreatic carcino-

genesis in experimental animals (see Refs. [40,94] for

reviews). For example, elevation of serum CCK (either by

injection of CCK, by feeding with protease inhibitors, or by

surgical manipulation) stimulated the development of aza-

serine-induced preneoplastic lesions in rat pancreas, and the

effect was partially blocked by the CCK-1 receptor-selective

antagonist CR 1409 [40]. In contrast, elevation of serum

Gamide by treatment with the protein pump inhibitor

lansoprazole did not significantly increase azaserine-in-

duced preneoplastic pancreatic lesions in the rat model

[95]. The development of ductular carcinoma in hamster

pancreas following treatment with N-nitrosobis(2-oxopro-

pyl)amine does not appear to be enhanced by CCK [40], or

reduced by CCK-1 receptor antagonists [96,97].

The role of the CCK-2 receptor in pancreatic neoplasia

has also been investigated in mouse models. A transgenic

mouse strain expressing the CCK-2 receptor in the exocrine

pancreas was created by fusing the elastase I promoter to the

human CCK-2 receptor gene [98,99]. Pancreatic weight in

these mice was increased by 40% over wild-type controls

[99,100]. Malignant transformation was observed in 3/20

(15%) homozygous crosses between this strain and a strain

expressing Gamide in pancreatic h-cells [100].

10. Role of gastrins in development and progression of

colorectal cancer

Early data suggested that some colorectal cancers and

cell lines synthesised gastrin and expressed gastrin recep-

tors, although the percentage of positive tumours varied

greatly between groups (Table 1). Moreover, the growth of

some colorectal carcinoma cell lines was stimulated by

exogenous Gamide, and could be inhibited by gastrin

receptor antagonists (see Ref. [2] for review). These obser-

vations were most commonly explained in terms of an

autocrine or paracrine loop. Reports of hypergastrinaemia

in patients with colorectal cancer also raised the possibility

that gastrin might act as an endocrine proliferative agent,

with the source of gastrin remaining undefined.

Reports of increased circulating Gamide concentrations in

patients with colorectal carcinomas compared to controls

have generated considerable controversy.Many of the reports

did not take into account the presence or absence ofH. pylori,

a known cause of hypergastrinaemia. Similar uncertainty

exists over the effect of tumour resection on Gamide concen-

trations [101–103]. In contrast, two studies, both of which

were controlled for H. pylori status, have reported that serum

concentrations of gastrin precursors are elevated in patients

with colorectal cancer [87,104]. A single study has reported

that colon cancer resection results in a reduction in the serum

concentrations of gastrin precursors, but not Gamide [105].

The results of these studies are consistent with the hypothesis

that colorectal cancers produce progastrin but are deficient in

the ability to process progastrin to Gamide [87].

More recent data support the view that Gamide and the

CCK-2 receptor do not play a significant role in the growth of

the majority of colorectal carcinomas. Reubi et al. [74] found

by receptor autoradiography that CCK-2 receptors were

expressed in only a small subset (4%) of colorectal carcino-

mas, and cautioned that the presence of receptors in nonma-

lignant tissue ‘‘contaminating’’ the tumour could give rise to

an overestimate of receptor-positive tumours. A recent study

confirmed that CCK-2 receptor mRNAwas detected in only a

A. Aly et al. / Biochimica et Biophysica Acta 1704 (2004) 1–106

subset (38%) of colorectal carcinoma specimens by PCR, and

localized the receptor to both cancer cells and polymorpho-

nuclear cells in the lamina propria by immunohistochemistry

[22]. In animal models, hypergastrinaemia induced by fun-

dectomy failed to produce increased tumour rates [106,107].

After treatment with azoxymethane, transgenic mice over-

expressing Gamide had no more aberrant crypt foci (prema-

lignant lesions) [108], or subsequent tumours [114], than

wild-type controls. Rats treated with the CCK-2 receptor

antagonist CR2945 had fewer dimethylhydrazine-induced

colorectal tumours than control at 38 weeks after exposure

[109], but at week 52 there was no difference [110]. When

taken together with the infrequent occurrence of both Gamide

peptide production and CCK-2 receptor expression in human

colorectal carcinomas, the animal data suggest that a role for

Gamide may be limited to a small subset of tumours.

A mis-spliced variant of the CCK-2 receptor has been

described in colorectal carcinoma [111]. The variant, which

retains intron 4, constitutively increased intracellular Ca2+

and cell growth. Although the original publication reported

that the variant was expressed in 8/8 colorectal carcinomas,

but not in adjacent normal tissue, a German group was

unable to detect variant mRNA in 79 colorectal carcinoma

specimens by PCR [22]. The variant mRNA was also

observed in pancreatic cell lines and in pancreatic tumours,

but not in the surrounding healthy tissue [112,113].

Recent studies focussing on progastrin and Ggly confirm

that it may be these peptides rather than Gamide that play a

role in colorectal carcinogenesis. The possible tumour-pro-

moting effects of gastrin precursors have been studied in

transgenic mice treated with carcinogens. In mice transgenic

for a human gastrin minigene containing the human gastrin

promoter, human gastrin mRNA was detected from embry-

onic day 18 to adult [36]. Progastrin expression was

restricted to the liver, but the animals had markedly in-

creased concentrations of progastrin in serum; other circu-

lating forms of gastrin were either decreased or unchanged.

After treatment with azoxymethane, the transgenic mice had

increased numbers and sizes of tumours compared to wild-

type mice. In addition, an increased proportion (42%) of

tumours in the progastrin group was in the proximal colon

compared to controls where the majority was in the distal

colon [114]. These observations correlated well with find-

ings of increased numbers of aberrant crypt foci in the

progastrin group in an earlier study [108]. Even short-term

exposure (4 weeks) of azoxymethane-treated rats to exog-

enous Ggly resulted in a significant increase in the number

of aberrant crypt foci formed [38]. The observation that

exogenously administered Ggly [38] or endogenous trans-

genic production of progastrin [37] can potentially promote

colorectal cancer by increasing the number of aberrant crypt

foci suggests that early activation of the gastrin gene may

similarly provide a tumour-enhancing environment via an

autocrine pathway. A surprising recent observation by

Singh’s group [115] that gastrin-deficient mice developed

more colonic adenocarcinomas than wild-type mice after

treatment with azoxymethane was interpreted in terms of an

inhibitory role for Gamide in colorectal carcinogenesis. This

interpretation is consistent with the proposal that it is the

non-amidated forms of gastrins that are responsible for the

acceleration of colon carcinogenesis.

While gastrin precursors appear to act as growth factors

in established colorectal cancer, their role in the early

stages of colorectal cancer development needs further

investigation. Of potentially great interest is the recent

finding that gastrin gene expression in the colon may be

related to activation of h-catenin. Mutations in the APC

tumour suppressor gene responsible for the Familial Ade-

nomatous Polyposis syndrome occur with high frequency

in sporadic colon cancer, and lead to abnormal interactions

with h-catenin, a protein involved in anchorage of inter-

cellular junction structures. When released from its an-

choring role, h-catenin also seems to be involved in

nuclear transcription of a variety of oncogenes. Mutations

in the APC gene result in such a process and can be

thought of as activating h-catenin. Koh et al. [116] have

demonstrated that, in mice heterozygous for a mutation in

the APC gene (APCmin), the gastrin gene is a downstream

target of activated h-catenin. When coupled with findings

that hypergastrinaemia promotes adenoma progression in

APCmin mice [117], that crosses between APCmin mice

and mice ubiquitously overexpressing Ggly under the

control of the metallothionein promoter have more polyps

[116], and that crosses between APCmin mice and gastrin

gene knockout mice have fewer polyps [116], this obser-

vation suggests that gastrin gene activation may represent

an early event in colorectal carcinoma pathogenesis. Gas-

trin gene expression is also induced by the k-ras oncogene,

which has been implicated in colorectal cancer pathogen-

esis [118].

In summary, these findings strongly suggest that colonic

neoplastic tissue consistently synthesises progastrin but is

deficient in the processing of progastrin to Gamide. The

degree of processing is quite variable between individual

tumours. Activation of the gastrin gene in colonic mucosa

cells may occur when carcinoma develops, as a result of

mutations in the APC, h-catenin or k-ras genes. As dedif-

ferentiation is frequently a feature of neoplastic transforma-

tion, such activation may represent regression toward a less

differentiated cell type. It is interesting that gastrin precur-

sors are found in high concentration in developing embry-

onic colon tissue whereas Gamide is not [119]. It may be

that gastrin precursors are important growth factors in

colonic development and that colon cancer and gastrin are

linked ontogenically.

11. Therapeutic implications

The conflicting nature of the reports on the presence and

source of hypergastrinaemia in patients with colorectal

cancer suggests that measurement of circulating gastrin

A. Aly et al. / Biochimica et Biophysica Acta 1704 (2004) 1–10 7

concentrations will not be a useful diagnostic tool, although

it is known that an elevated serum gastrin confers a nearly

fourfold increased risk of developing colorectal cancer [120].

However, the observation that antiserum against gastrin

[121] or against the CCK-2 receptor [122] inhibits hepatic

invasion of human colorectal carcinoma cell lines suggests

that treatment with such antisera may be a future treatment

option in some cases. In fact, immunization with a conjugate

between gastrin and diphtheria toxin has now passed phase II

trials in patients with advanced pancreatic cancer [123].

The development of [111In]-labelled gastrin/CCK deriv-

atives has opened new opportunities for the diagnosis and

treatment of tumours expressing the CCK-2 receptor. Pilot

experiments with [111In]-diethylenetriamine-pentaacetic ac-

id-d-Glu(1) minigastrin in nude mice bearing xenografts of a

medullary thyroid carcinoma [124], and in 45 patients with

metastatic medullary thyroid carcinoma [125], have given

promising results. Similar data have been obtained with

[111In]-diethylenetriamine-pentaacetic acid-desSO4-CCK8

[126]. The use of these compounds in gastrointestinal cancer

is an exciting prospect, although it should be recalled that

only a minority of gastrointestinal tumours will contain the

CCK-2 receptor.

An alternative approach may involve the use of gastrin

receptor antagonists. Studies in gastrin knockout animals

demonstrating a reduction in tumour incidence suggest that

gastrin receptors may provide an additional target for

prophylaxis or therapy for colorectal cancer. In animal

models, treatment with a CCK-2 receptor antagonist results

in only a slight reduction in tumour incidence [109,110].

Progress in this area will be critically dependent on the

identification and characterisation of the receptors involved

in mediating the effects of the gastrin precursors in colon

cancer, and on the development of selective antagonists

against them.

Recent studies suggest that a potential target of amidated

gastrin is cyclooxygenase-2 (COX-2), the enzyme involved

in the conversion of arachidonic acid to prostaglandins

[127,128]. COX-2 is overexpressed in colorectal cancers,

and nonsteroidal anti-inflammatory drugs which inhibit

COX-2 reduce the mortality from colorectal cancer [129].

Although it has been established that gastrin stimulates COX-

2 expression, and that inhibition of COX-2 reverses the

trophic effect of gastrin on colorectal carcinoma cells, these

observations have been confined to cells that express the

CCK-2 receptor [127,128]. Since only a small minority of

colorectal cancers is CCK-2 receptor-positive, the protective

mechanism by which nonsteroidal anti-inflammatory drugs

reduce the risk of developing colorectal cancer is likely to be

mainly independent of the action of amidated gastrin.

12. Conclusion

There seems little doubt that amidated gastrins and

gastrin precursors play a role in gastric and colorectal cancer

pathogenesis, respectively. Thus, the majority of colorectal

cancers demonstrate an active gastrin gene and produce

gastrin precursors to which they may well respond mito-

genically. Whether gastrin precursors and their receptors

may be a therapeutic target should now become the focus of

continuing work in this area.

Acknowledgements

This work was supported in part by the Austin Hospital

Medical Research Foundation, by the Royal Australian

College of Surgeons (Reg Worcester Fellowship to A.A.),

and by grants 114123 (to A.S.) and 208926 (to G.B.) from

the National Health and Medical Research Council of

Australia.

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