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Role of Glucagon-Like Peptide-2 in Rodent Models of Colon Cancer
by
Shivangi Trivedi
A thesis submitted in conformity with the requirements for the degree of Master of Science
Graduate Department of Physiology University of Toronto
© Copyright by Shivangi Trivedi (2011)
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Role of Glucagon-Like Peptide-2 in Rodent Models of Colon Cancer Master of Science Shivangi Trivedi Graduate Department of Physiology University of Toronto 2011
Abstract of thesis
Glucagon-like peptide-2 (GLP-2) is an intestinotrophic and intestinal anti-
inflammatory hormone. Hence, I hypothesized that treatment with degradation-
resistant hGly2GLP-2 increases, while blocking endogenous GLP-2 decreases
colorectal cancer (CRC) in rodents. In mice, treatment with dextran sodium sulphate
(DSS) and azoxymethane (AOM) induced colitis-associated CRC, which was further
increased by treatment with hGly2GLP-2 and reduced by blocking endogenous GLP-2
with the antagonist hGLP-23-33. Moreover, while colonic damage scores (CDS) was
not altered by hGly2GLP-2 or hGLP-23-33 treatment, hGly2GLP-2 increased small
intestinal growth and hGLP-23-33 reduced jejunal crypt cell proliferation. In rats fed
with of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and high fat (HF)
diet for aberrant crypt foci (ACF) induction, treatment with hGly2GLP-2 increased
small intestinal growth and ACF occurrence. Moreover, in rats fed with PhIP-HF diet
for tumour induction, early treatment with hGly2GLP-2 appears to increase the
occurrence of intestinal tumours. Collectively, these findings indicate a pro-
carcinogenic role for both exogenous and endogenous GLP-2.
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Acknowledgements
First and foremost, I would like to thank my supervisor, Dr. Brubaker who has been a
constant source of guidance, support and encouragement during all my time at the
Brubaker lab. I am grateful for being a student of such an inspiring and learned
scientist who has helped me grow not only as a student, but also as a human being. I
will be forever indebted to my parents Jaydev and Jayshree for their limitless
patience, and unconditional support for my education. I would also like to thank
many of the past and present members of the Brubaker lab – people who changed my
life for the better. Ms. Katherine Rowland, for being an amazing mentor under whose
guidance I did my undergraduate project. Her infectious optimism, guidance and
friendship are invaluable. I am grateful to Stuart Wiber for his summer and project
work that contributed to my thesis. I thank Drs. Roman Iakoubov and Lina Lauffer
for being such incredible role models with their ability to link their encyclopedic
clinical knowledge to basic research and Dr. Victor Wong, for showing me the lighter
side of research. I thank Monika Poreba and Andrew Mulherin for their
companionship and help throughout the past two years. I appreciate the technical help
and guidance that I have received from our laboratory technician Angelo Izzo and
Leila Tick for taking great care of my research animals. Lastly, I would like to thank
my committee members Dr. Adria Giacca, Dr. Young-In Kim and Dr. Freda Miller
for their feedback and guidance throughout the course of this research project. I feel
that I have immensely benefited as a student and as a person from my time at the
Brubaker lab. I am grateful for the opportunities to inquire and learn that I have
gained during this time and would like to carry this same curiosity and scientific
attitude in my future endeavours.
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Table of Contents
Abstract of thesis ……………………………………………………………………………………….(ii) Acknowledgements …………………………..……………………………………………………….(iii) Table of contents ……………………………………………………………………………………… (iv)List of figures ……………………………………………………………………………………………(vi) List of abbreviations …………………………………………………………………………………(vi) 1. Introduction ................................................................................................................... 1
1.1 Rationale .................................................................................................................. 1
1.2 Glucagon-like peptide-2 (GLP-2) .......................................................................... 2
1.2.1 Synthesis ............................................................................................................ 2
1.2.2 Other proglucagon derived peptides .................................................................. 2
1.2.3 Secretion ............................................................................................................ 4
1.2.4 Degradation and clearance ................................................................................. 5
1.2.5 GLP-2 receptor, location and signaling ............................................................. 6
1.2.6 Biological functions of GLP-2 ........................................................................... 6
1.2.7 The functional mediators of GLP-2 ................................................................... 8
1.3 Inflammatory bowel disease and GLP-2............................................................. 10
1.3.1 Animal models of IBD ..................................................................................... 11
1.3.2 Current treatments for IBD and the potential for GLP-2 ................................. 15
1.3.3 IBD and cancer ................................................................................................ 17
1.4 Colorectal cancer .................................................................................................. 18
1.4.1 Familial and sporadic CRC .............................................................................. 18
1.4.2 Animal models of familial and sporadic CRC ................................................. 20
1.4.3 IBD-associated carcinogenesis ........................................................................ 25
1.4.4 IBD-associated cancer models ......................................................................... 26
1.4.5 GLP-2 and colon cancer ................................................................................... 28
1.5 Rationale, hypothesis and specific aims ................................................................. 30
2. Materials and Methods ............................................................................................... 32
2.1 Animals .................................................................................................................. 32
2.2 Experimental protocols with mice ....................................................................... 32
2.2.1 DSS-colitis pilot study ..................................................................................... 32
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2.2.2 Colonic damage score (CDS) ........................................................................... 34
2.2.3 AOM-DSS study .............................................................................................. 34
2.2.4 Morphometry ................................................................................................... 35
2.2.5 Immunohistochemistry (IHC) .......................................................................... 36
2.3 Experimental protocols with rats ........................................................................ 37
2.3.1 PhIP-ACF study ............................................................................................... 37
2.3.2 PhIP-tumour study ........................................................................................... 38
2.3.3 Histopathology ................................................................................................. 40
2.4 Statistical analysis ................................................................................................. 40
3. Results .......................................................................................................................... 42
3.1 DSS-colitis pilot study ........................................................................................... 42
3.3.1. DSS doses of 1.0-3.0% were used to establish a chronic murine model of colitis with no associated mortality in our animal facility. ....................................... 42
3.2 DSS-AOM study .................................................................................................... 45
3.2.1 Administration of h(Gly2)GLP-2 or antagonism of endogenous GLP-2 with hGLP-23-33 administration does not alter body weight.............................................. 45
3.2.2 Administration of hGly2GLP-2 increases small intestinal growth .................. 45
3.2.3 GLP-2 does not increase colon growth or chronic colitis damage................... 513.2.4 Administration of hGly2GLP-2 and hGLP-23-33 may alter high-grade dysplasia and colon cancer incidence ....................................................................... 55
3.2.5 Administration of hGly2GLP-2 or hGLP-23-33 does not alter the number of DCAMKL-1-positive stem cells in the colon ........................................................... 55
3.3 PhIP-ACF study .................................................................................................... 58
3.3.1 Feeding AIN-93G diet with PhIP reduces rat body weight ............................. 58
3.3.2 Administration of hGly2GLP-2 increases small intestinal but not colon growth ....................................................................................................................... 58
3.3.3 hGly2GLP-2 administration increases the number of ACF in the colon ......... 61
3.3.4 GLP-2 does not alter MDF occurrence ............................................................ 62
3.4 PhIP-tumour study ............................................................................................... 65
3.4.1 Administration of hGly2GLP-2 does not alter total amount of PhIP consumed or body weight in rats .............................................................................. 65
3.4.2 Administration of hGly2GLP-2 during an early phase of cancer development may lead to increased intestinal tumour burden……………………………………………………………………………..……….......…………….65
4. Discussion..................................................................................................................... 68
References ........................................................................................................................ 77
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List of Figures
1.1 Proglucagon processing………………………………………………………………………………3 1.2 Schematic representation of the mechanism of action of GLP-2……………………….92.1 Protocol for DSS colitis pilot………………………………………….………………………….33 2.2 Protocol for DSS-AOM study…………………………………………………………………….33 2.3 Protocol for PhIP-ACF study……………………………………………………………………..39 2.4 Protocol for PhIP-tumour study……………………………………………………...…………..39 3.1 DSS-colitis pilot study: Body weight, colon weight, length and weight to length ratio……………………………………………………………………………………………………………..43 3.2 DSS-colitis pilot study: Colonic damage score……….…………………………………….44 3.3 DSS-AOM study: Body weights…………………………………….………………………….. 47 3.4 DSS-AOM study: Body weights on day of sacrifice……………………………………..48 3.5 DSS-AOM study: Small intestinal weights………………………………..…………………49 3.6 DSS-AOM study: Jejunal crypt-villus lengths and proliferation..………….…………50 3.7 DSS-AOM study: Colon weights………………………………………………………………..52 3.8 DSS-AOM study: Colon lengths……………………………………………….………………..53 3.9 DSS-AOM study: Colonic damage score and proliferation…………….………………54 3.10 DSS-AOM study: Colonic dysplasia and cancer…………………………………………56 3.11 DSS-AOM study: DCAMKL-1 stem cells…………………………………………………57 3.12 PhIP-ACF study: Body weights………………………………………………………………..59 3.13 PhIP-ACF study: Small intestinal and colon growth……………………………………60 3.14 PhIP-ACF study: ACF occurrence………………………..…………………………………..63 3.15 PhIP-ACF study: MDF occurrence…………………………….……………………………..64 3.16 PhIP-tumour study: Total PhIP fed and body weight……………………..……………66 3.17 PhIP-tumour study: Occurrence of suspected intestinal tumours……………..……67
List of Tables
1.1 IBD and selected animal models of collitis: a summary…………………………………12 1.2 CRC and selected animal models: a summary……………………………………………...22 1.3 Selected IBD-associated CRC models: a summary……………………………………….27
List of abbreviations:
A Adenine (nucleotide) ACF Aberrrant crypt foci AOM Azoxymethane APC Adenomatous polyposis coli C Cytosine (nucleotide) cAMP Cyclic adenosie monophosphate CD Crohn’s disease CRC Colorectal cancer DCAMKL-1 Doublecortin and calmodulin-kinase-like-1
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DMH 1,2-Dimethylhydrazine DNA Deoxyribonucleic acid DPP-IV Dipeptidyl peptidase-IV DSS Dextran sulphate sodium EGF Epidermal growth factor ErbB/EGF-R Epidermal growth factor receptor G Guanine (nucleotide) GLP-1 Glucagon-like peptide-1 GLP-2 Glucagon-like peptide-2 GLP-2R Glucagon-like peptide-2 receptor GLUT2 Glucose transporter GPCR G-protein coupled receptor IBD Inflammatory bowel disease IGF-1 Insulin-like growth factor-1 IGF-1R Insulin-like growth factor-1 receptor IL Interleukin iPGDP Intestinal proglucagon-derived peptides KGF Keratinocyte growth factor KRAS Kirsten ras MDF Mucin depleted foci min Multiple intestinal neoplasia NOD2 nucleotide-binding oligomerization domain-containing 2 (NOD2) PhIP 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine PI3K Phosphoinsotiol-3 kinase SGLT Sodium glucose transporter T Thymine (nucleotide) TNBS Trinitrobenzene sulphonic acid TNF-α Tumour necrosis factor-α UC Ulcerative colitis Wnt Wingless
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1. Introduction
1.1 Rationale
According to the Canadian Cancer Society, an estimated 22,500 Canadians were
diagnosed with cancer of the colon and rectum and approximately 9100 died due to it in
the year 2010. The consumption of red and processed meats is a known risk factor for the
development of CRC, as are agents such as animal fats and sugars (www.cancer.ca, [1]).
These findings suggest that humoral factors that link diet and intestinal growth may have
a role in the development of intestinal carcinogenesis. One such factor is GLP-2, a 33-
amino acid intestinotrophic hormone that is secreted from the enteroendocrine L-cell in
response to ingestion of nutrients, including fats and sugars in particular [2; 3; 4]. While
the physiological action of GLP-2 leads to intestinal regrowth after fasting and refeeding,
the pharmacological actions of GLP-2 include intestinal mucosal growth, increased
digestion and absorption, improved blood flow and decreased inflammation [2; 5; 6; 7; 8;
9; 10; 11]. Hence, a long-acting GLP-2 analog (teduglutide, Gattex™) is in clinical trials
for the treatment of short bowel syndrome and inflammatory bowel disease
(www.clinicaltrials.gov). However, data from our lab have demonstrated that GLP-2 also
increases colonic preneoplastic lesions and, possibly, cancer in a mouse model of
carcinogenesis induced by a chemical carcinogen. As the risk of developing CRC can be
as high as 18% after 30 years of developing inflammatory bowel disease (IBD) it is
essential to study the potential carcinogenicity of GLP-2 in a model of IBD [12].
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1.2 Glucagon-like peptide-2 (GLP-2)
This section will provide a brief overview of the synthesis, secretion, metabolism
and actions of GLP-2.
1.2.1 Synthesis
GLP-2 is synthesized as part of the 160-amino acid long proglucagon protein,
which is expressed in pancreatic α-cells, intestinal L-cells and the central nervous system
[13; 14]. Proglucagon undergoes tissue-specific processing to produce a number of
peptides involved in energy metabolism and gut homeostasis. In the pancreatic α-cell,
proglucagon undergoes cleavage by the enzyme prohormone convertase 2 (PC2) to
produce glicentin-related pancreatic peptide, glucagon, intervening peptide-1 and the
major proglucagon fragment, which contains glucagon-like peptide-1 (GLP-1) and GLP-
2 [15; 16; 17]. In contrast, the enzyme prohormone convertase-1/3 cleaves proglucagon
in the intestinal L-cell and possibly the brain to liberate glicentin, oxyntomodulin,
intervenening peptide-2, GLP-1 and GLP-2 (Figure 1.1) [15; 16; 17].
1.2.2 Other proglucagon derived peptides
A majority of the peptides co-secreted with GLP-2 have biological functions. In
brief, GLP-1 is an incretin, increasing glucose-dependent insulin secretion [18].
Moreover, GLP-1 also reduces gastric emptying and food intake. The hormone glicentin
is known to have modest intestinal growth and incretin effects [2; 19], while
oxyntomodulin is mostly known for its effects to induce satiety and reducing gastric acid
secretion [20]. Since glicentin/oxyntomodulin, GLP-1 and GLP-2 are produced in
equimolar quantities within the L-cell, results from studies examining the secretion of
either of these peptides are applicable to the secretion of GLP-2. Hence, in the following
NC
PC2
Pancreas: PC2
Glicentin-related
pancreatic peptide
Glucagon Major proglucagonfragment
Oxyntomodulin
PC1/3
Intestinal L cell: PC1/3
Glicentin GLP-17-37/36NH2 GLP-2
ProglucagonProglucagon
Figure 1.1 Proglucagon processing. Proglucagon undergoes post-translational processing by the prohormone convertaste enzyme PC2 in the panreatic islet and and by PC1/3 in the intestinal L-cell to produce a number of proglucagon-derived peptides.
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section, a brief description of the secretion of these intestinal proglucagon-derived
peptides (iPGDPs) is outlined.
1.2.3 Secretion
The intestinal L-cell that secretes iPGDPs is found throughout the small and large
intestine, with greatest density in the distal ileum and colon [13]. The L-cell is ‘open’ to
the lumen of the intestine, exposing it to the luminal contents of the gut [13]. Moreover, it
is also in contact with the vasculature and innervated by the parasympathetic nervous
system [21; 22; 23]. Hence, secretion of the iPGDPs can be modulated by nutrients,
neuronal and humoral factors. Release of the iPGDPs is also pulsatile throughout the day
with the pulse amplitude increasing after meal ingestion in a biphasic fashion [24]. The
first phase of secretion occurs within 15-30 min of meal ingestion, and a second phase
occurs at 60-90 min [4]. Notably, the ingestion of pure glucose or lipids but not protein
increases secretion of iPGDPs [4]. As a majority of orally ingested glucose is absorbed
before it reaches the distal gut, fat is likely to be the most physiologically relevant L-cell
secretagogue.
An interesting conundrum in the study of GLP secretion has been that orally
ingested nutrients do not reach the distal gut till 60-90 min after meal ingestion, while the
first phase of secretion occurs within 15-30 min. Studies with muscarinic antagonists,
duodenal ligation and vagotomy models have demonstrated that the first phase of
nutrient-induced iPGDP secretion is mediated by the vagus nerve [24; 25]. The second
phase of secretion is due to direct L-cell contact with nutrients in the lumen of the
intestine. In addition, hormonal signals such as glucose-dependent insulinotropic peptide
and insulin have also been shown to increase iPGDP secretion [26; 27]. Lately, studies
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examining the effect of various iPGDP secretagogues have also gained vogue due to an
enhanced interest in the incretin effect of GLP-1. As such, the effects of agonists for
various G-protein coupled receptors (GPCR) such as GPR 119 and TGR5 in inducing L-
cell secretion have also been studied [28; 29; 30; 31]. Additionally, it has been shown
that in rat models of surgical resection of the bowel, there is an increased amount of
iPGDP secretion [32]. Thus, while, the main physiological stimulus for L-cell secretion is
nutrient ingestion, there are a number of additional factors through which the L-cell can
be stimulated to release the iPGDPs, including GLP-2.
1.2.4 Degradation and clearance
The endogenous bioactive form of GLP-2, (GLP-21-33) has an Alanine at position
2 [33]. As such, it undergoes degradation by the widely-expressed enzyme, dipeptidyl
peptidase IV (DPP-IV) to produce the degradation product GLP-23-33 [33; 34; 35].
Moreover, GLP-2 also undergoes renal clearance, making the half-life of GLP-21-33 in the
circulation a short 7 minutes [36]. Hence, blocking the action of DPP-IV by using DPP-
IV inhibitors such as sitagliptin also results in an increase in the levels of endogenous
bioactive GLP-2 [37]. Indeed, in humans with IBD, the level of GLP-21-33 is increased in
comparison to normal subjects, in part due to reduced DPP-IV activity [38]. However,
degradation-resistant human GLP-2 analogs with Glycine at postion 2 (Gly2GLP-2) are
utilized for in a majority of the studies examining the pharmacological actions of GLP-2
[34]. Finally, GLP-23-33 is a partial agonist of the GLP-2 receptor (GLP-2R) that can act
as an antagonist at low doses. Hence, in experimental settings, GLP-23-33 may be used as
an agent to block the actions of endogenous GLP-2 [5].
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1.2.5 GLP-2 receptor, location and signaling
The GLP-2 receptor (GLP-2R) is a 7-transmembrane domain GPCR that shares
sequence homology with the glucagon and GLP-1 receptors, making it a member of the
glucagon-secretin receptor superfamily [39]. In humans, the GLP-2R is found on
chromosome 17 and has recently been implicated by a genome wide association study in
the regulation of erythrocytes in patients with sickle cell anemia [39; 40].
The GLP-2R has been found on enteroendocrine cells, enteric neurons and
subepithelial myofibroblasts throughout the gut, with the highest expression in jejunum,
followed by the duodenum, ileum and colon [39; 41; 42; 43]. Moreover, when radioactive
125I-GLP-2 was injected in rodents, it had maximum localization in the small intestine,
but was also found in the colon [44]. The GLP-2R is also expressed at very low levels in
the central nervous system, mainly in the dorsomedial hypothalamus, in addition to other
extrahypothalamic regions [41].
In cells transfected with the GLP-2R, treatment with GLP-2 results in activation
of the cyclic adenosine monophosphate (cAMP) and mitogen-activated protein kinase
pathways, consistent with its coupling with Gαs protein [39; 41]. However, activation of
both cAMP and Akt-dependent pathways has been observed in cells that naturally
express the GLP-2R, including intestinal subepithelial myofibroblasts [45].
1.2.6 Biological functions of GLP-2
GLP-2 has a number of gut-specific effects that will be discussed in detail in this
section. However, it is notable that the action of GLP-2 on the central and enteric nervous
systems decreases food intake and gastric motility [46; 47; 48]. Moreover, GLP-2 has
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also been shown to increase hipbone mineral density, possibly by improving intestinal
calcium absorption [49].
Following observation of massive growth in small intestinal diameter and
absorptive surface area of a patient with a glucagon-secreting endocrine tumour, it was
postulated that glucagon or a related peptide had intestinotrophic actions [50]. This factor
was shown to be GLP-2, when Drucker et al. demonstrated that GLP-2 administration in
mice increased small bowel growth [2]. GLP-2 increases small intestinal crypt and villus
length, crypt cell proliferation and mucosal surface area [2; 51; 52]. GLP-2 has also been
shown to increase colonic weight and crypt cell proliferation to a modest extent [42; 52].
In addition, GLP-2 has been shown to increase small and large bowel length and to
reduce apoptosis [10; 51; 53]. Studies in mice administered with GLP-2 have shown
increased expression of digestive enzymes, resulting in improved digestive capability [7].
Moreover, in rat models GLP-2, increases the expression of the sodium-glucose
transporter, SGLT-1, and glucose transporter, GLUT2, to improve absorptive function [8;
54]. Finally, GLP-2 has also been shown to increase intestinal (mesenteric) blood flow in
both experimental animal models and humans [55; 56; 57; 58]. As a result of this
improvement in both mucosal growth and digestive capability, treatment with GLP-2
improves nutrient absorption. At a molecular level, treatment with GLP-2 has been
shown to increase the action of the pro-proliferative and anti-apoptotic Wingless (Wnt)-
β-catenin and phosphatidyl inositol-3 kinase (PI-3K)-Akt pathways, through both insulin-
like growth-1 (IGF-1) and epidermal growth factor (EGF) receptor (EGF-R/ ErbB)-
dependent mechanisms [6; 34].
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1.2.7 The functional mediators of GLP-2
As noted earlier, in the intestinal epithelium, the GLP-2R is found only on
enteroendocrine cells, which comprise of less than 5% of total epithelial cells [41].
Moreover, the GLP-2R is also found on enteric neurons and subepithelial myofibroblasts
[42; 59] (see schematic, Figure 1.2). However, the major target of the actions of GLP-2 is
the epithelial cell compartment of the crypts and villi [2; 34]. This apparent paradox has
been explained by the discovery of a number of endocrine and paracrine factors that
mediate the actions of GLP-2. For the physiological and pharmacological mucosal
growth effects of GLP-2, IGF-1, ErbB receptor and its ligands and well as keratinocyte
growth factor have been implicated [6; 42; 52; 60; 61]. For increase in intestinal blood
flow, endothelial nitric oxide synthase mediates the effects of GLP-2 [58; 62].
Furthermore, the hormone vasoactive intestinal polypeptide is known to mediate the anti-
inflammatory effects of GLP-2 [59].
Studies examining the physiological actions of GLP-2 have also demonstrated
mucosal growth functions of GLP-2. In mice, fasting reduces small intestinal weight,
crypt and villus lengths as well as crypt cell proliferation, while refeeding induces
mucosal regrowth. Making use of the GLP-2 antagonist GLP-23-33 to block actions of
endogenous GLP-2, Shin et al demonstrated in mice that endogenous GLP-2 is essential
for inducing the regrowth of the intestine in response to refeeding after a fast [5].
Similarly, Bahrami et al demonstrated that the refeeding-induced mucosal regrowth
phenomenon was absent in mice lacking the GLP-2R [6]. Moreover, in rats also, the
intestinal mucosal regrowth after fasting and refeeding has been shown to be, in part,
GLP-2-dependent [63]. Thus, due to the increased mucosal growth, digestion and
Enteric neuron:NOS, VIP
Goblet cells
Vasculature
L-cell
GLP-2RCrypt
Villus
Subepithelial myofibroblasts:IGF-1, KGF, ErbBligands
Stem cells
Figure 1.2 Schematic representation of the mechanism of action of GLP-2. In the intestine, the GLP-2R is present on enteroendocrine cells, including the L-cell, subepithelial myofibroblasts, and enteric neurons. The activation of GLP-2R on the subepithelial myofibroblasts leads to the release of growth factors such as IGF-1, KGF and ErbBligands. The actions of these growth factors ultimately lead to increased epithelial proliferation. Moreover, activation of the GLP-2R on enteric neurons leads to the release VIP, which has anti-inflammatory effects.
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absorption functions of GLP-2, a long-acting, degradation-resistant analog of GLP-2
(hGly2GLP-2, teduglutide, Gattex©) is now awaiting Food and Drug Administration
approval for the treatment of parenteral nutrition-dependent short bowel syndrome
patients. Moreover, due to the anti-inflammatory actions of GLP-2 as discussed below,
teduglutide is also in Phase II clinical trials for the treatment of Crohn’s disease
(www.clinicaltrials.gov).
1.3 Inflammatory bowel disease and GLP-2
In addition to its trophic effects on the normal bowel, GLP-2 is an effective agent
for reducing mucosal injury and the severity of inflammation in rodent models of IBD
[10; 11; 59; 64]. A more in-depth introduction to IBD, the pathophysiological changes
involved, and the effect of GLP-2 administration in models of IBD is herein presented.
IBD refers to conditions that involve chronic or intermittent inflammation of the
bowel, mainly Crohn’s disease (CD) and ulcerative colitis (UC). In Canada, an estimated
200,000 individuals live with IBD (www.ccfc.ca). Although not completely
indistinguishable, CD and UC normally differ by the location and extent of inflammation.
Patients with CD may have inflammation in discrete parts of the gastrointestinal tract –
ranging from the mouth to the anus, often called “skip lesions” that may develop in
patches [65]. However, in patients with UC, the inflammation involves only the colon,
starting distally and developing proximally in a continuous manner. Moreover, the extent
of inflammation in CD is not limited to the mucosa and can be transmural, while the
inflammatory lesions are mostly mucosal in UC. Thus, inflammation in the colon can be
a result of either UC or CD of the colon (Crohn’s colitis) [65]. In general, CD and UC are
chronic diseases, with sporadic periods of active intestinal inflammation called disease
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“flares” that may be separated by a few days to a few decades. During periods of disease
flares, patients exhibit a number of symptoms including diarrhea, intestinal spasms, blood
in stools, and abdominal pain (www.ccfc.ca).
1.3.1 Animal models of IBD
CD and UC involve inflammation of the gastrointestinal tract that is thought to be
a result of the interaction between genetic traits and environmental factors. Genome-wide
association studies have linked genes involved in mediating innate immunity, autophagy
and inflammatory responses with the occurrence of CD and UC [65; 66]. Evidence of
genetic linkage is more comprehensive for CD than UC. Hence, studies in twins have
also shown that while 58.3% of monozygotic twins have concordance in developing CD,
there is only 6.3% concordance for UC [67; 68]. In addition, other, non-genetic traits
such as dietary components, antibiotic use and the diversity of gut flora have also been
implicated in the pathogenesis of IBD [69; 70; 71; 72].
In the past, there was little convincing evidence for a direct causal link between
genetic and environmental factors in IBD etiology. Hence, research with animal models
of IBD largely relied on using the cytotoxic agents dextran sodium sulfate (DSS, MW
40,000-50,000), trinitrobenzene sulfonic acid (TNBS), the polysaccharide carrageenan
and intestinal microbial infection (see summary of selected models in Table 1.1) [73; 74;
75; 76]. For models of CD involving the small bowel, TNBS is the preferred agent, since
it causes inflammation in the part of the intestine where it is administered. DSS and
carrageenan selectively cause inflammation in the colon, starting with the distal-most
part, while TNBS induces colonic inflammation if infused rectally [59]. Oral gavage is
required for microbial infection models of IBD (eg. Citrobacter rodentium infection).
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Table 1.1 IBD and selected animal models of collitis: a summary
Species Location Induction Similarities to human
CD Human
Can be skip lesions from mouth to anus; can be transmural Spontaneous N/A
Ulcerative colitis Human
Colon; mostly mucosal
Spontaneous (typically develops in second or third decade of life) N/A
Carrageenen Rodent, rabbit
Cecum, colon, rectum Inducible
Weight loss, diarrhea, blood and mucous in stools; in colonic, cecal, rectal mucosa: cellular infiltrates into the lamina propria, crypt abcesses and ulceration
DSS Rodent Colon Inducible
Weight loss, blood in stools, diarrhea; in colonic mucosa: crypt shortening, crypt abecesses, crypt erosion, crypt distortion and epithelial hyperproliferation
IL-10-/- Mouse
Duodenum, proximal jejunum, proximal colon
Spontaneous (develops at 4-8 wk age)
Lower body weight, anemia; in intestinal mucosa: inflammation, distortion of crypt architecture, crypt branching, hyperproliferation
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Citrobacter rodentium infection Mouse Colon Inducible
Body weight loss; in the intestinal mucosa: CD3+cell infiltrates in colonic lamina propria and epithelium, mucosal thickening, crypt cell hyperplasia
NOD2-/- Mouse Colon Inducible (using DSS)
Weight loss, loose stools with blood; normal intestinal mucosa without DSS treatment
TNBS Rodent Intestine Inducible
Weight loss, diarrhea, rectal prolapse; in the intestinal mucosa: transmural inflammation, infilitration of T cells, increased inflammatory cytokines and crypt cell hyperproliferation
The resulting citrobacter rodentium infection, inflammation and hyperplasia are primarily
colonic [77]. It should be noted that the susceptibility to these agents depends on the
mouse strain and the microbial colonization of the gut, which can vary with the animal
facility. Among these agents, DSS is the most widely studied and will be discussed in
further detail here.
In general, DSS disrupts gut homeostasis by cytotoxicity of intestinal epithelial
cells that separate innate immune cells from the contents of the intestinal lumen [73; 78].
The disruption of the intestinal epithelial barrier, leading to increased intestinal
permeability leads to an innate immune response mediated by intestinal lymphocytes [79;
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80; 81; 82; 83]. The resulting intestinal inflammation has IBD-like pathology, which
resembles human UC [73]. Notably, colitis induction by DSS occurs even in germ-free
mice, leading to the conclusion that the disruption of epithelial mucosa by DSS is
sufficient for intestinal inflammation [84]. Moreover, it is known that intestinal
inflammation in DSS-colitis is due to innate immunity because even mice with severe,
combined immunodeficiency and lacking adaptive immunity (B-cells and T-cells)
develop DSS-colitis [85; 86]. Moreover, DSS can also be used to selectively establish
either acute (single 5-7 d DSS treatment followed by euthanasia) or chronic (intermittent
DSS 5-7 d treatments, separated by periods of recovery) models of colitis [73; 87]. DSS
is also a useful agent in studies examining genetic models of IBD, as discussed below.
In 2001, two groups simultaneously published results linking the occurrence of CD in
human beings with variations in the NOD2 gene, which encodes the nucleotide-binding
oligomerization domain-containing 2 (NOD2) protein that is involved in modulating
innate immune responses for gut homeostasis [88; 89]. In the following years, as the
variants of other genes linked to IBD were found, a number of genetic mouse models of
IBD were developed. In general, IBD in humans has been linked with variations or
mutations in genes that encode proteins involved in maintenance of the intestinal barrier,
recognition of gut microbes, autophagy and innate immunity [65; 90; 91; 92; 93; 94; 95].
However, mouse models with variations or mutations in these genes have limited success
at establishing relevant models of IBD. For example, while NOD2-/- mice develop a
colitis response to DSS, they do not develop spontaneous colitis or Crohn’s disease [96].
Moreover, while other genetic models of colitis such as IL-10-/- mice may develop
spontaneous colitis, the colitis development is not synchronized or completely
15
reproducible in every facility [97]. Thus, although genetic models of IBD are effective in
the elucidation of pathways involved in IBD pathology, they still require IBD-induction
and their reproducibility is dependent on the animal facility. Hence, DSS alone or in
combination with genetic manipulation remains an excellent model for studying UC
because of its reproducibility, feasibility and similar pathology to human UC.
1.3.2 Current treatments for IBD and the potential for GLP-2
Since both CD and UC are chronic diseases that present with varying intensity
and sporadic periods of inflammation, a general definitive cure for IBD does not exist.
Complete remission may be achieved by surgical removal of inflamed parts of the
intestine, although this can lead to inefficient nutrient absorption. In general, IBD
treatment comprises of two main categories – 1) therapies for management of IBD
symptoms without altering gut inflammation and 2) therapies aimed at reducing
inflammation within the gut. Therapies that reduce the severity of symptoms associated
with IBD include antidiarrheals, antispasmodics and analgesics. Additionally, IBD
treatment also includes therapies that reduce inflammation of the gut by blocking the
formation of inflammatory mediators, such as sulfasalazine and 5-aminosalicylic acid,
corticosteroids or other immunomodulators such as methotrexate and even antibiotics
(www.ccfc.ca, [98]. These therapeutics, however, may not be specific to the intestine and
are associated with a number of side effects. Hence, the development of biological agents
that target known inflammatory pathways that are active in IBD has gained vogue.
Following the successful use of agents that block the actions of tumour necrosis factor-α
(TNF-α) such as infliximab and etanercept, other biological therapeutics targeted towards
the inflammatory pathways involved in IBD have been developed and are in various
16
stages of clinical testing [99]. Moreover, analogs of growth factors such as growth
hormone, IGF-1, EGF and GLP-2 have been suggested as potential therapeutics for the
treatment of IBD, since these agents contribute towards growth of the intestinal mucosa,
improve nutrient absorption and may reduce inflammation [100; 101],
www.clinicaltrials.gov.
GLP-2 is a particularly interesting potential therapeutic for IBD treatment due to
its success at being an intestine-specific growth factor that also improves digestion and
absorption and has anti-inflammatory effects [2; 7; 8; 9]. The effects of GLP-2 on disease
activity associated with IBD have been evaluated in animal models of IBD. Drucker et al.
used a model of acute colitis induced by DSS to show that administration of hGly2GLP-2
concurrent with DSS treatment led to reduced body weight loss, improved survival,
greater intestinal mucosal area and integrity [10]. Similarly, L’Heureux et al also
demonstrated that hGly2GLP-2 treatment improved survival, small intestinal growth, and
crypt cell proliferation in a murine model of acute colitis induced by DSS. Treatment
with hGly2GLP-2 also reduced structural colitis damage and inflammatory
myeloperoxidase activity in this model [11]. In addition, GLP-2 has been effective in
reducing disease activity and microscopic intestinal damage DSS-induced colitis as well
as in models of colitis and ileitis and colits induced by TNBS [59]. Interestingly, in these
studies, the administration of hGly2GLP-2 decreased both crypt cell proliferation and
apoptotic indices in the inflamed part of intestine [59]. Moreover, it was also found that
GLP-2 exerts anti-inflammatory actions by reducing expression of proinflammatory
cytokines, such as interferon-γ, TNF-α and IL-1β and increasing expression of the anti-
inflammatory cytokine IL-10. It was found that these functions of GLP-2 are dependent
17
on the action of vasoactive intestinal polypeptide secreted from enteric neurons of the
submucosal plexus [59]. Moreover, studies of colitis in IL-10-/- mice, which are a chronic
colitis model, have shown that the anti-inflammatory actions of GLP-2 do not require IL-
10 [64]. Thus, a number of studies investigating the effects of GLP-2 in mostly acute
models of colitis and ileitis, have defined the anti-inflammatory function of GLP-2
suggesting it as a possible therapeutic for IBD treatment. Hence, a degradation-resistant
analog of GLP-2 is currently in clinical trials for treatment of active CD (Phase II).
Published studies with small cohorts of CD patients indicated that treatment with
hGly2GLP-2 does have the beneficial effect of inducing remission and improving
mucosal healing [102]. However, further studies that examine the long-term effects of
GLP-2 in chronic models of IBD are required, especially considering the close links
between IBD and CRC.
1.3.3 IBD and cancer
The relative risk for developing intestinal cancer in patients suffering from IBD is
much greater than the general population – 4.5 relative risk for developing CRC with
colitis and 33.2 for developing small intestinal cancer with CD involving the small bowel
[103]. The CRC risk of patients with Crohn’s colitis is similar to those with UC and the
presence of primary sclerosing cholangitis in these patients further increases the CRC risk
[104; 105; 106; 107]. Moreover, while the risk of developing CRC for the general
population is negligible before 50 years of age, IBD patients are diagnosed with CRC at
younger ages of 40-45 years [12; 108]. The probability of developing CRC for UC
patients increases by 1-2% per year after having UC for 10 years, with up to 18% disease
risk at 30 years [12]. Moreover, CRC associated with IBD is the cause of death for almost
18
one sixth of patients with IBD [12]. Thus, the cancer risk and mortality associated with
IBD-linked intestinal cancer is substantial and is linked to the type, duration and location
of the disease. Hence, if GLP-2 is to be a safe therapeutic for patients, its long-term
effects in the context of increased cancer risk due to IBD must be evaluated.
1.4 Colorectal cancer
Cancer of the colon and/or rectum occurs as result of aberrant cancerous lesions
that are most commonly of an epithelial or glandular origin (adenocarcinoma). Since the
gut epithelium is a major target of GLP-2, a detailed review of the types of CRC, their
pathogenesis, models of CRC and the currently existing literature on the role of GLP-2 in
CRC models is discussed below.
1.4.1 Familial and sporadic CRC
Since the pathogenesis and etiology of CRC greatly varies, depending on genetic
and environmental factors as well as co-morbidities with other diseases, CRC is broadly
classified as inherited (familial), sporadic when it is a result of random genetic mutations
in the colon, or “inflammation-associated” when it is a result of co-morbidity with IBD
[109; 110].
An estimated 20-30% of CRC is considered to be a result of familial inheritance [110;
111]. However, <5% of CRC cases are a result of known inherited mutations [112]. The
first known gene to be directly linked with CRC occurrence was adenomatous polyposis
coli (APC), a tumour suppressor [113; 114; 115]. An autosomal dominant mutation in
APC is present in individuals with a condition known as familial adenomatous polyposis,
who comprise of <1% of all CRC cases [116]. These individuals develop multiple
aberrant benign growths, or polyps, in their colons, which may develop into carcinoma if
19
not removed. Moreover, an estimated ~2 % of individuals with CRC are known to have
an inherited condition called hereditary nonpolyposis colorectal cancer (Lynch
syndrome), which involves heterozygous mutations in mismatch repair (MMR) genes
(MSH2, MLH1, PMS2 and MSH6) [117; 118; 119; 120; 121]. These genes encode
enzymes that are required for correcting errors in DNA replication or repair. Most Lynch
syndrome patients lose heterozygosity in these genes primarily in the colon, and later in
other epithelial tissues. In the colon, this normally results in development of a single large
tumour with characteristic “microsatellite instability” – erroneous repeats of small DNA
sequences [118]. Other familial cancers include MYH-associated polyposis,
hamartomatous polyposis syndromes and hyperplastic syndromes that comprise of less
than 1% of all CRC cases [116]. The cases of familial syndromes of CRC, thus, comprise
of a very small percentage of all CRC patients. However, their study has provided a
wealth of information about the causal links between genetic aberrations and cancer, and
the interplay of genetics and environment in sporadic CRC pathogenesis.
The initial discovery of inherited APC mutations as causes of cancer were
followed by findings that upto 80% sporadic human adenocarcinomas of the colon had
presence of APC mutations [113; 122; 123; 124]. Following this discovery, the role of the
pro-proliferative and anti-apoptotic Wnt-β-catenin signaling pathway that is essential for
normal intestinal homeostasis but is also involved in the development of CRC, was
elucidated [125; 126]. It is now well recognized that the transcription factor β-catenin is
normally present at low levels in the cytosol and along the cellular membrane. On
activation of Wnt signaling, it translocates into the nucleus and exerts its pro-
proliferative, anti-apoptotic, and anti-differentiation effects through transcription of genes
20
such as cmyc and sox9 [61; 127]. In the absence of active Wnt signaling, β-catenin binds
to a degradation complex made up of the APC, glycogen synthase kinase-3β (GSK-3β)
and casein kinase to be phosphorylated and targeted for degradation [125; 128]. Hence,
mutations in either the tumour suppressor APC or proto-oncogene β-catenin are linked to
the initiation of carcinogenesis of the colon [129; 130; 131]. In addition, growth factor
signaling pathways involving growth factor receptors that are active in the intestinal
epithelium such as IGF-1R and downstream PI3K as well as the EGF receptor and
Kirsten ras (Kras)/Raf pathways are linked to colon carcinogenesis initiation and/or
progression. [132; 133; 134; 135; 136; 137; 138].
1.4.2 Animal models of familial and sporadic CRC
Findings from human data about the molecular players in the development of
CRC have helped to better understand the pathogenesis of CRC. However, to study CRC
in experimental settings, the use of animal models is required. To study the effects of
various pro- or anti- carcinogenic factors, studies with animal models of CRC use
carcinogenic agents or genetic mutations to induce cancer (summarized in Table 1.2).
The most commonly used agents that specifically target the intestinal mucosa are
methylating agents such as dimethylhydrazine (DMH) and its metabolite azoxymethane
(AOM) [139]. In addition, heterocyclic amines such as 2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine (PhIP), aromatic amines such as dimethyl-4-
aminobiphenyl (DMAB) and alkylynitrosamide compounds such as N-methyl-N-
nitrosourea (MNU) are known colon carcinogens [139; 140; 141; 142]. In general, these
carcinogens cause somatic mutations in genes involved in pathways that are implicated in
human CRC, including disruption of the Wnt-APC-β-catenin axis, inducible nitric oxide
21
synthase, cyclooxygenase-2 as well as mutations in the pro-oncogenic Kras gene [136;
143; 144; 145; 146; 147; 148]. Although there are reports of extra-intestinal
carcinogenicity for AOM and DMH, their carcinogenic action is mainly limited to the
colon [139; 149]. Other carcinogens, however, have widely reported extra-intestinal
carcinogenic actions. For example, cancer of the prostate and mammary gland for
heterocyclic amine PhIP, cancer of the prostate and pancreas by DMAB, while MNU is a
carcinogen normally used for inducing widespread DNA damage, including gametic
mutations [142; 150; 151; 152; 153; 154; 155]. Moreover, since AOM is a metabolite of
DMH and is closer to the ultimate carcinogenic species in addition to being safer to
handle, it is the preferred carcinogen to DMH [140; 141]. The carcinogenic actions of
AOM and PhIP only are discussed here, since AOM-induced cancer is specific to the
colon and PhIP is a common dietary carcinogen, making them highly relevant to the
development of human sporadic CRC [139; 140; 142; 156; 157].
When injected systemically, AOM is metabolized in the liver to form the DNA-
reactive metabolite methylazoxymethanol, which requires further metabolism by the P-
450 enzyme CYP2E1. The resultant reactive species leads to the methylation of guanine
nucleotides at the O6 position, leading to G:CA:T transitions [139; 140; 144] .
Mutagenesis by AOM targets the APC, β-catenin and Kras genes, in particular due to the
presence of mutational “hot spots” in these genes [123; 144; 158; 159; 160]. The reason
for the colon-specific action of AOM is unknown although, the colonic enzymatic
activation or transport of AOM/MAM through bile acids to the colon has been
hypothesized [141]. A limitation with the use of AOM is that although the genetic
22
mutations it causes are similar to those of human sporadic cancer, in nature, AOM is a
rare dietary carcinogen that is found in cycad flour [139].
Table 1.2 CRC and selected animal models: a summary
Type Typical age Molecular mechanism
Similarities to human
Human
FAP
Development of multiple intestinal polpys as early as teenage years (average age 39 years)
Mutations in APC gene, leading to development of a number of benign colonic polyps that become cancerous N/A
Lynch syndrome 35-50 years
Mutations in MMR genes N/A
Sporadic >50 years
Mutations found progressively in APC, Kras and p53; leading to dysregulated Wnt and growth factor signaling, decrease in cell cycle arrest and apoptosis N/A
Genetic models
APCmin+/-
60 days, survive up to 120 days
Heterzygous loss of APC gene
Mutation in same gene as FAP patients, but tumours predominantly in distal small intestine instead of colon
DNA mismatch gene mutations (Eg. Msh2-/- and Msh6-/-)
Mice develop spontaneous tumours without requring chemical induction. Majority of Msh2-/- mice die in 6-8 months, Msh6-/- die within 18 months
Mutations in DNA mismatch repair genes
DNA repair defects similar to human. However, mice heterzygous for mutations in Msh2 or Msh6 do not develop tumours.
23
Sporadic models
DMH
Induced, typically in adult rodents
Most commonly, mutations in APC, β-catenin, Kras in the colonic epithelium
Mutations in APC and Kras, as seen a majority of sporadic human CRC (ie. dysregulated Wnt signaling)
AOM (metabolite of DMH)
Induced, typically in adult rodents
Most commonly, mutations in APC, β-catenin, Kras in the colonic epithelium
Mutations in APC and Kras, as seen a majority of sporadic human CRC (ie. dysregulated Wnt signaling)
PhIP/HF diet
Induced, typically in adult rodents
Most commonly, mutations in APC, β-catenin, but not Kras in the colonic epithelium
Cancer induced by PhIP, a common heterocyclic amine found in human diet (i.e. in cooked meat); cancers induced by PhIP have dysregulated Wnt signaling with mutations in APC
Contrary to the rare availability of AOM in human diet, PhIP is a common dietary
carcinogen that is found in cooked meat and fish [142; 156; 161]. Hence, PhIP is an
attractive agent for inducing cancer in rodent models because of its high relevance to the
human diet. Indeed, PhIP has been used to establish working models of preneoplastic
lesions and large intestinal tumour induction that are widely used [162; 163; 164; 165;
166; 167; 168]. Similar to AOM, PhIP requires metabolic activation by liver enzymes to
form N-hydroxylated metabolites, which form PhIP-DNA adducts [157]. The actions of
PhIP ultimately result in mutations to the APC and β-catenin, but not Kras genes [162;
165; 169; 170; 171]. Although cancer-induction by PhIP treatment is not completely
intestine-specific, a cancer induction protocol by Ubagai et al that employs intermittent
24
PhIP-feeding with high fat (HF) diet containing trans-fatty acids favours the induction of
large intestinal tumours [162]. Hence, the PhIP-HF diet model is also useful for studying
the effects of various agents on cancer induction.
In addition to chemically induced mutagenesis, animal models of CRC with
inactivating mutations in tumour suppressor genes or activating mutations in proto-
oncogenes are also quite common. Among these, a popular model for the study of CRC is
the Apcmin/+ mouse, which has a heterozygous mutation in the APC gene, rendering it
with a multiple intestinal neoplasia (min) phenotype [172]. Although, these mice have a
mutation in the same gene as familial adenomatous polyposis patients, they develop
multiple tumours throughout the intestine, with a majority of the neoplastic lesions in the
distal small intestine instead of the colon [172; 173]. Moreover, these mice require great
care perinatally, start developing anemia at 60 days, and are normally expected to live
only for120 days [172]. In addition, the tumours in these mice have high levels of Wnt
signaling, which may render them less useful for studying factors that modulate Wnt
signaling in the initiation stages of CRC [174; 175]. Hence, although, the Apcmin/+ mouse
model is widely used, the main limitations associated with its use are the viability of the
mice and location of the tumours. Moreover, mice with variations in the ApcMin/+
phenotype, β-catenin and mitogen-activated protein kinase pathway are also available for
the study of colon cancer[126; 176; 177]. Finally, for the study of tumours involving
microsatellite instability, mice mutant in mismatch repair genes have also been developed
[178; 179; 180]. Thus, studies of familial human CRC etiology have contributed to a
better understanding of sporadic CRC, and the development of animal models of CRC
has been useful for evaluation of pro- and anti-cancer agents.
25
It should be noted that although a number of animal models of cancer have been
developed, the cost and time required for colon cancer induction is often prohibitive.
Hence, a number of surrogate biomarkers of carcinogenesis that predict the pro- or anti-
carcinogenic effects of agents within shorter time periods have been discovered.
Preneoplastic lesions that fall between the “epithelial hyper-proliferation” and “early
adenoma” steps of carcinogenesis in Fearon and Vogelstein’s stochastic model of
carcinogenesis have been described. They include, aberrant crypt foci (ACF) that can be
visualized in the colonic tissue as retaining dark methylene blue staining, with larger
diameters and protruding surfaces, representing crypts with increased proliferation [181].
Moreover, β-catenin accumulated crypts have been identified by immunohistochemistry
and detection of crypts with high levels of β-catenin indicating increased pro-oncogenic
Wnt signaling [182]. Mucin-depleted foci (MDF) that have been identified by the lack of
mucin staining represent crypts with loss of goblet cell function that are presumably
progressing towards de-differentiation [183; 184; 185].
1.4.3 IBD-associated carcinogenesis
The current understanding of the links between IBD and CRC includes the role of
chronic inflammation as a cancer-promoting factor in the development of dysplastic
lesions that advance towards carcinoma. Hence, the molecular mechanisms involved in
developing IBD-associated cancer are a sequence of mutations of tumour suppressors and
proto-oncogenes distinct from the one described by Fearon and Volgelstein for sporadic
CRC [186; 187]. It is postulated that chronic inflammation leads to the formation of
reactive oxygen and nitrogen species, causing oxidative damage to cellular components
[188]. Oxidative damage, may then lead to mutations in genes encoding DNA mismatch
26
repair enzymes such as MLH1, MSH2, MSH6, or PMS2, leading to further aberrations in
epithelial cell cycling and growth [189]. Moreover, genomic instability is found in almost
all cases of IBD-associated cancer, with both chromosomal instability and microsatellite
instability detected in dysplasia as well as cancer associated with IBD [189; 190; 191].
Mutations in genes encoding tumour suppressors such as APC, p53 and KRAS are also
found in cases of cancer-associated with IBD [192]. However, the sequence of
mutagenesis differs from sporadic CRC, in that p53 mutations are observed first in the
preliminary stages of carcinogenesis as opposed to later stages, as seen in sporadic cancer
[186; 192]. Moreover, pro-inflammatory cytokines such as TNFα and IL-6 are known to
promote tumour cell growth/survival through nuclear factor-κB and Stat3-mediated
signaling [109; 193; 194; 195; 196; 197].
1.4.4 IBD-associated cancer models
The following section will discuss the available models of IBD-associated cancer,
including the dextran sodium sulfate-azoxymethane (DSS-AOM) model of colitis
associated with CRC (summarized in Table 1.3). In general, models of chronic colitis can
be considered models of IBD-associated cancer [87; 198]. Hence, a number of genetic
models of colitis are good candidate models for studying IBD-associated cancer. For
example, as noted above, mice that lack the anti-inflammatory cytokine IL-10 (IL-10-/-)
develop adeonocarcinomas [198; 199]. However, inflammation development in these
mice depends on the animal facility, which may lead to differential effects on
carcinogenesis [97]. Mice with a null mutation in the Stat3 gene in macrophages also
develop chronic inflammatory bowel disease with spontaneous inflammation-associated
adenocarcinoma development [200]. Similar results are seen in mice lacking the MUC2
27
gene, which are deficient in mucin production and develop chronic colitis due to faulty
intestinal barrier function [201; 202]. However, as these models of genetic alterations
leading to chronic colitis and carcinoma are less widely used as they are difficult to
synchronize, a more widely used approach for colitis-associated cancer induction,
therefore, employs the cytotoxic agent DSS with or without AOM [87; 141; 203]. Models
of chronic colitis can be established by intermittent cycles of DSS and recovery.
Table 1.3 Selected IBD-associated CRC models: a summary
Species Induction Mechanism of carcinogenesis
Similarities to human
DSS-AOM Rat, mouse Inducible
Oncogenic mutations induced by AOM, chronic inflammation by three cycles of DSS
Tumour induction protocol involves intermittent DSS administration, mimicking human chronic colitis, tumours predominantly in distal colon
Multiple DSS cycles Mouse Inducible
Chronic colonic inflammation leading to flat dysplasia/cancers and dysplasia associated with lesions or masses
Histopathology similar to human, formation of both flat and polypoid dysplasia, increase in dysplasia or cancer occurrence with longevity of disease
IL-10-/- Mouse
Spontaneo-us or inducible with AOM
IL-10 deficiency leads to chronic inflammation through increase in Th1 cytokines - IL-2, interferon-ϒ, TNF-α and IL-6
Chronic colitis that increases severity with aging, leading to carcinogenesis. In the colon, adenocarcinomas with back-to-back growth of glands and loss of nuclear polarity
28
Macrophage-specific Stat3-/- Mouse
Spontaneo-us
Increase in mammalian target of rapamycin-Stat3 signaling in epithelial and tumour cells
Spontaneous development of colonic inflammation and tumour lesions in areas of inflammation.
MUC2-/- Mouse Spontaneo-us
Increased migration and proliferation, decreased apoptosis of epithelial cells; increase in cmyc expression in tumour cells
Colitis starting at 5 wk of age, increasing in severity with age. Tumours found in large intestines of older (1 yr) mice. Invasive carcinomas of epithelial origin.
As Cooper et al demonstrate, 1-4 cycles of DSS followed by 14-120 d recovery
periods can lead to development of dysplasia or cancer in Swiss Webster mice [87].
However, these effects may be strain-specific and the high DSS concentration required
(5% w/v) may cause animal welfare issues. A more robust method for the induction of
colitis-associated cancer was developed by Tanaka et al, using AOM (10 mg/kg) for
cancer induction and one week of low-dose (2% w/v) DSS for colitis induction, followed
by 20 wk recovery for induction of colitis-associated cancer or dysplasia [203]. This
protocol was later modified by Neufert et al to include one AOM injection and three
cycles of DSS and recovery for colitis-associated cancer induction within 10 wk [141].
This model of colitis-associated cancer is now widely used, with modifications, as it has a
high success rate of adenocarcinoma development, with good reproducibility, feasibility
and fewer animal welfare issues [197; 204]. Hence, for my studies, this DSS-AOM model
of colitis-associated cancer was utilized.
29
1.4.5 GLP-2 and colon cancer
The literature examining the potential effect of GLP-2 in carcinogenesis is not
extensive. One of the earliest studies was performed even before the discovery of GLP-2
as a mucosal growth factor in the intestine. Hence, in a model of surgical jejunal
resection in male Wistar rats treated with AOM, it was found that the numbers of
duodenal tumours correlated with the amount of enteroglucagon immunoreactivity,
indicative the levels of iPGDPs [32]. This correlative study can be considered to be an
early indication of the potential carcinogenic effects of GLP-2. However, to specifically
examine the role of GLP-2 in colon cancer, Thulesen et al used female C57BL mice with
repeated injections of DMH, followed by a 2-month recovery, as a model [205]. They
found that both native GLP-2 and the degradation-resistant analog, hGly2GLP-2,
increased the number of colonic neoplasms in this model, suggesting a role for exogenous
GLP-2 in driving colon cancer growth. However, a limitation of this study was its focus
on the effects of GLP-2 in cancer growth or promotion, rather than initiation.
Furthermore, none of the tumours were cancerous; possibly as the animals were
terminated before progression could occur [205]. A study by Koehler et al also examined
the role of GLP-2 in carcinogenesis, albeit in very different models. They found that in
nude mice on BALB/C background, GLP-2 treatment does not alter the growth of
xenografts of colon cancer cells (SW480 and DLD-1 cell lines) transfected with the gene
for GLP-2R. GLP-2 also does not alter the growth of intestinal tumours in C57BL/6J-
Apcmin/+ mice [206]. Moreover, this study also demonstrated that colon cancer cell lines,
such as DLD-1, SW480, SW48, SW620, SW116, Caco-2, HT29, Colo201, Colo205, and
Colo320, do not express the GLP-2R. Furthermore, when transfected with GLP-2R, the
30
SW480, DLD-1 and HT29 cell lines do not demonstrate a growth response to GLP-2
treatment [206]. In contrast, a study by Masur et al found GLP-2R expression on the
SW480 and HT29 cell lines and demonstrated that GLP-2 in conjunction with DPP-IV
inhibition increased the proliferation and migratory activity of these cells in vitro [207].
Given our knowledge of the indirect mechanism through which GLP-2 exerts its effects,
the role of GLP-2 in colon carcinogenesis should ideally be studied through in vivo
settings [208]. Furthermore, these studies did not elucidate the role of endogenous GLP-2
in modulating colon cancer initiation [205; 206; 207]. Hence, a study from the Brubaker
lab examined C57BL/6 mice treated with AOM and administered vehicle, early or late
hGly2GLP-2 or hGLP-23-33 to elucidate the roles of both exogenous and endogenous
GLP-2 in altering the initiation and promotion stages of murine colon cancer [209].
Interestingly, this study demonstrated that, while treatment with hGly2GLP-2 increased
the occurrence of colonic preneoplastic lesions – ACF and MDF, blocking the actions of
endogenous GLP-2 reduced the number of ACF [209]. Furthermore, the effects of early
vs. late treatment with hGly2GLP-2 were similar in increasing ACF and MDF numbers.
Finally, tumours were found in only 10% of the mice, all hGly2GLP-2-treated [209].
Thus, the literature examining the effects of GLP-2 on colon cancer demonstrates varied
findings.
Comparing reports on the actions of exogenous and endogenous GLP-2 on colon
cancer in the literature is difficult, given the paucity of intestinal cancer models that have
been studied. Hence, to draw conclusions regarding the broader applicability of the
results from these studies, it becomes essential to examine the effect of GLP-2 in
additional models of colon cancer. Moreover, considering the cancer-promoting effect of
31
IBD on colon cancer, the actions of both exogenous and endogenous GLP-2 should be
examined on colon cancer growth in this condition.
1.5 Rationale, hypothesis and specific aims
GLP-2 is a hormone that increases intestinal growth, potentially increasing
carcinogenic risk within the colon. However, it also reduces intestinal inflammation,
possibly reducing the risk for colon carcinogenesis. As such, it is uniquely positioned as a
potential pro- or anti-carcinogenic agent in the colon, depending on the presence of other
associated risk factors. Moreover, the possibility of blocking endogenous GLP-2 action
as an approach to reduce colon cancer occurrence also remains an attractive avenue for
research. Hence, I hypothesized that treatment with hGly2GLP-2 increases, while
blocking endogenous GLP-2 decreases colon cancer growth in rodent models of colonic
dysplasia and cancer. My aims were therefore, to assess whether i) in a murine model of
murine colitis-associated cancer induced by DSS and AOM, hGly2GLP-2 increases,
while hGLP-23-33 decreases colon cancer incidence; ii) in a rat model of colon ACF
induced by PhIP and HF diet feeding, treatment with hGly2GLP-2 increases, while
treatment with hGLP-23-33 decreases ACF occurrence and iii) in a rat model of large
intestinal tumours induced by cycles of intermittent PhIP and HF diet feeding,
hGly2GLP-2 administration increases tumorigenesis at both the intiation and post-
initiation stages of tumour development.
32
2. Materials and Methods
2.1 Animals
Adult (6-10 wk), male C57BL/6 mice and adult (5-6 wk), male Fischer 344 (F344)
rats were purchased from Charles River Canada (Charles River Canada, St. Constant,
Québec, Canada). All experimental protocols were approved by the Animal Care
Committee of the University of Toronto. All animals were housed in a facility with a 12
hour light-day cycle and given ad libitum access to water and food.
2.2 Experimental protocols with mice
2.2.1 DSS-colitis pilot study
Morbidity associated with increasing doses of dextran sulfate sodium salt (DSS)
alone, to establish a model of chronic colitis was determined by making modifications to
the colitis-associated cancer protocol described in [141]. Briefly, adult, male C57BL/6
mice were provided with 1-3% DSS (MW 40,000-50,000, USB Corporation, Cleveland,
Ohio, USA) in their drinking water for 1 wk followed by 2 wk of recovery (Figure 2.1).
This cycle was performed three times. During the DSS cycles, mice were monitored daily
for signs of morbidity including weight loss, dehydration and blood in stools. At the end
of 9 weeks, the mice were sacrificed and whole colons were fixed in 10% neutral-
buffered formalin for histological analyses. Formalin-fixed colons were embedded in
paraffin and 4-μm cross sections of the proximal, middle and distal part of the colon were
prepared at the pathology laboratory at University Health Network.
◊ 6-10 wk old, male C57BL/6 mice:◊ DSS: 2.5% (w/v)◊ Injections (sc, bid): Vehicle, 1.5 μg h(Gly2)GLP-2, 30 ng hGLP-23-33
Set 1: Mice injected with vehicle, h(Gly2)GLP-2, or hGLP-23-33 (n=8-10)Set 2: Mice injected with vehicle or h(Gly2)GLP-2 (n=18-19)Set 3: Mice injected with vehicle or hGLP-23-33 (n=7-20)
DSS
AOM injection
InjectionsDSS DSS
1 wk1 wk2 wk 2 wk 2 wk1 wk
DSS DSS DSS
1 wk1 wk2 wk 2 wk 2 wk1 wk1 wk
Figure 2.1 Protocol for DSS colitis pilot study.
Figure 2.2 Protocol for DSS-AOM study.
◊10 wk old, male C57BL/6 mice:◊ DSS: 1.0, 2.0, 2.5, 3.0 % (w/v)
33
34
2.2.2 Colonic damage score (CDS)
Digital images of H&E-stained sections of the colon were obtained using a Zeiss
AxioPlan microscope (Carl Zeiss Canada, Don Mills, Ontario, Canada) with AxioVision
4.8 software. As described previously [11; 210], one cross-section from each segment of
the colon was then analyzed for colonic damage. Colonic injury was quantified by
employing a grading system whereby the loss of the bottom one-third of crypts was
categorized as Grade 1 damage, the loss the bottom two-thirds was identified as Grade 2
damage and loss of the entire crypt structure was categorized as Grade 3 damage in a
blinded fashion. The areas of damage were measured using AxioVision software and
expressed as a proportion of the total mucosal area (1=10% up to 10=100% of mucosal
area) and were multiplied with their respective grade of damage to obtain a composite
CDS. A composite CDS was the average of damage in the three segments.
2.2.3 AOM-DSS study
To determine the effect of GLP-2 in a model of murine colitis-associated cancer, a
modified protocol from [141] and [197] was used. Adult (6-10 wk), male C57BL/6 mice
(Charles River) were injected with azoxymethane (AOM) (10 mg/kg, i.p.) (Sigma-
Aldrich Canada Ltd., Oakville, Ontario, Canada) and allowed to recover for 1 wk (Figure
2.2). At the beginning of wk 2, they were given 2.5% DSS in drinking water for 1 wk,
followed by 2 wk of recovery. This cycle was performed three times. During the DSS
cycles, mice were monitored daily for signs of morbidity, dehydration and blood in
stools. Rodent chow mash, mixed in regular water was provided in cases of severe
dehydration. Mice were weighed weekly and were randomized to one of three treatment
groups after the last day of DSS treatment. During the last cycle of recovery, mice were
35
injected with either vehicle (50 mM ammonium bicarbonate, 200 μL, sc, bid), hGly2GLP-
2 (1.5 μg, 200 μL, sc, bid) (long acting analog, American Peptide Company, Sunnyvale,
California, USA) or hGLP-23-33(30 ng, 200 μL, sc, bid) (partial agonist of GLP-2
receptor, American Peptide Company). Mice were sacrificed at the end of the protocol.
On the day of sacrifice, the mice were administered with vehicle, hGly2GLP-2 or hGLP-
23-33 according to their respective groups, 3 h prior to time of sacrifice [209]. Small
intestinal weight, colon weight and colon length were obtained after gentle cleaning.
Sections (0.5-2 cm in length) from the jejunum (5-10 cm proximal from the mid-small
intestine), ileum (5-cm proximal from the ileocecal valve) and colon were either frozen
on dry ice or fixed in 10% neutral buffered formalin for analyses. Any tumours found
were photographed, fixed in formalin and if possible, frozen on dry ice for storage at -80
°C. Because of the large number of animals involved in this study, this experiment was
performed using three separate cohorts over a 12 month period. The three cohorts of mice
were Set 1 (all 3 treatment groups; n=8-10), Set 2 (vehicle and hGly2GLP-2 treatments
only; n=18-19) and Set 3 (vehicle and hGLP-23-33 only; n=9-17).
2.2.4 Morphometry
Morphometry for crypt depth and villus height was performed on digital images
obtained with a Zeiss AxioPlan microscope. The AxioVision software was used to
measure crypt depth (distance from the bottom of the crypt to the crypt-villus junction)
for an average of 46 well-oriented crypts from H&E-stained jejunal sections in a blinded
fashion. The crypt-villus lengths were obtained by measuring the distance between the
bottoms of the crypts to villus tips for an average of 38 well-oriented crypt-villus units.
Mean villus height was obtained by subtracting mean crypt depth from the mean crypt-
36
villus length for each sample.
2.2.5 Immunohistochemistry (IHC)
Immunohistochemistry was performed on 4 μm cross-sections from the jejunum,
normal colon and tumour tissues. IHC for the proliferative marker Ki67 was performed
using a rat anti-mouse Ki67 antibody (1:150 dilution, Clone Tec-3, DakoCytomation,
Glostrup, Denmark), followed by visualization using a biotinylated mouse anti-rat
secondary antibody (Vector Laboratories, Burlingame, California, USA) with horseradish
peroxidase staining. Immunoreactivity was visualized using diaminobenzidine followed
by counter-staining with hematoxylin. For each specimen, 20-40 well-oriented crypts
from three cross sections of the jejunum or colon were selected for scoring in a blinded
manner. A positional analysis for Ki67 was performed by scoring cells from the crypt
bottom (position 1) up to position 20 on the crypt as positive or negative for Ki67 in a
blinded fashion.
Immunohistochemistry for the quiescent stem cell marker doublecortin and
calmodulin kinase-like-1 (DCAMKL-1) in sections from tumour tissues was performed
using an anti-human DCAMKL-1 C-terminal purified rabbit polyclonal antibody (1:30
dilution, Abgent, San Diego, California, USA). Cells positive for DCAMKL-1 were
counted and classified as belonging to normal, dysplastic or tumour tissues in a blinded
fashion. The total normal mucosal, dyplastic and tumour areas in each section were
quantified using AxioVision software and the number of DCAMKL-1-positive cells per
unit area of normal or tumour tissue was determined for each specimen as appropriate.
Immunohistochemistry for the canonical Wnt signaling marker β-catenin was performed
using a purified mouse anti-mouse β-catenin antibody (1:300 dilution, BD Transduction
37
Laboratories, Mississauga, Ontario, Canada) as described previously [61].
Immunohistochemistry for epithelial cytokeratins AE1/AE3 was performed using a
monoclonal mouse anti-human cytokeratin AE1/AE3 antibody (1:150 dilution, Dako
North America, Carpinteria, California, USA). A goat anti-mouse secondary antibody
(Vector Laboratories) followed by horseradish peroxidase treatment, diaminobenzidine
staining and hematoxylin counterstain was used to visualize the immunoreactivity. A
gastrointestinal pathologist blinded to the experimental treatments analyzed the H&E-
stained and AE1/AE3-stained tumour tissue sections as normal, high grade or low grade
dysplasia and cancer based on observations of structural damage and epithelial cell
invasion within the lamina propria or muscularis.
2.3 Experimental protocols with rats
2.3.1 PhIP-ACF study
To determine whether GLP-2 influences the development of dietary carcinogen-
induced preneoplastic lesions in rats, a modified protocol from [163] was followed
(Figure 2.3). In brief, 5-6 wk old, male F344 rats were given 400 ppm 2-Amino-1-
methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, Toronto Research Chemicals, North York,
Ontario, Canada) mixed in regular AIN93G powdered chow diet with 16.8% fat-derived
calories from soybean oil. (Dyets Inc., Bethlehem Philadelphia, USA) for two weeks.
Food intake and body weight were measured on alternate days during this period. This
was followed by 4 wk of high fat (HF) diet with 59.2% fat-derived calories, obtained by
supplementing AIN93G diet with Primex (hydrogenated vegetable oil, Dyets Inc.). In the
HF diet, 41.3% calories were derived from Primex and 17.9% from soybean oil. During
wk 3 and 4 of the study, the rats were injected with either 50 mM ammonium bicarbonate
38
(vehicle, 200 μL, sc, bid), hGly2GLP-2(40 μg, 200 μL, sc, bid, American Peptide Co.) or
hGLP-23-33 (2.5 μg, 200 μL, sc, bid, American Peptide Co.). During wk 5 and 6 of the
study, the doses of hGly2GLP-2 and hGLP-23-33 were increased to 60 μg and 3.75 μg
respectively to account for the increase in rat body weight. The rats were weighed twice
weekly during wk 3-6. At the end of wk 6, the rats were sacrificed and the whole small
and large intestines were removed, gently cleaned and weighed. The small intestine was
divided into 4 equal parts and sections from the duodenum, proximal jejunum, distal
jejunum and ileum were frozen on dry ice or fixed in formalin. Cleaned colons were
opened longitudinally, cut in three segments (proximal, middle and distal) and fixed flat
on Whatman paper between microscope slides in formalin for histological analysis.
Morphometry for jejunal crypt and villus length was performed as described above.
2.3.2 PhIP-tumour study
To determine the effect of GLP-2 on sporadic colon cancer, a model of rat colon
cancer was used, with modifications from [162] (Figure 2.4). Briefly, 6 wk old, male
F344 rats were given 400 ppm PhIP in regular AIN93G powdered chow diet for 2 wk
followed by 4 wk of HF diet. This cycle was repeated twice more, followed by 42 wk of
HF diet. The animals were divided into three groups; control, initiation and progression.
Animals in the control group received vehicle (50mM ammonium bicarbonate, 200 μL,
sc, bid) injections in wk 3-6 and 15-18. Animals in the initiation group received
h(Gly2)GLP-2 (40 μg in wk 3-4, 60 μg in wk 5-6, 200 μL, sc, bid) and vehicle in weeks
15-18. Animals in the progression group received vehicle during wk 3-6 and
h(Gly2)GLP-2 (80 μg in wk 15-16, 90 μg in wk 17-18, 200 μL, sc, bid) of the
experiment. Weekly body weights were recorded throughout the study.
42 wk4 wk4 wk4 wk 2 wk2 wk2 wk
PhIP PhIP
Injections: Vehicle or
h(Gly2)GLP-2
PhIP
Injections: Vehicle or
h(Gly2)GLP-2
4 wk2 wk
PhIP
Figure 2.3. Protocol for PhIP-ACF study.
Male, 5 wk old F344 rats
Injections: Vehicle Bicarb, h(Gly2)GLP-2 or hGLP-23-33
Figure 2.4 Protocol for PhIP-tumour study.
Male, 5 wk old F344 rats
39
40
The animals were also intermittently monitored for signs of colon cancer
development such as weight-loss and rectal bleeding and were sacrificed if deemed
necessary by veterinarians at the animal facility. All the remaining animals were
sacrificed in the 61st wk of the experimental protocol.
After sacrifice, the small intestine, cecum and large intestine were removed and
examined for signs of macroscopic lesions. Any macroscopic lesions were photographed.
The colons were then opened longitudinally, cleaned gently, cut in three segments and
fixed flat as described above.
2.3.3 Histopathology
To identify the preneoplastic lesions, aberrant crypt foci (ACF), longitudinally
opened colons were stained with a 0.05% (w/v) methylene blue for 1 min, and rinsed in
PBS. These colons were viewed under 5x and 10x objectives in a blinded manner to
identify single or groups of crypts with thicker and larger boundaries, darker staining and
seemingly protruding surfaces, as described previously [211]. To stain for mucin depleted
foci (MDF), the colons were rinsed in 70% ethanol, and treated with a high-iron diamine
solution (2.4 g/liter N-N-dimethyl-m-phenylene diamine, 0.4 g/liter N-N-dimethyl-p-
phenylene diamine, 1.68% ferric chloride) for 18 h followed by staining with Alcian blue
(1% Alcian Blue in 3% acetic acid) for 1 min and rinsing in 80% ethanol (modified from
previously described protocol [212]). Colons were scored for MDF in a blinded fashion,
using a 5x objective and were identified as single or clusters of crypts without mucin
staining.
2.4 Statistical analysis
All data are presented as mean ± SEM. For data from the DSS-colitis and DSS-
41
AOM mouse studies, statistical significance was established by performing 1-way
analysis of variance (ANOVA) followed by Bonferroni correction post hoc test as
appropriate (GraphPad Software, San Diego, CA). Data for all three sets of mice from the
DSS-AOM are not yet available. Where data from all mice are available, the cohorts were
combined, after analysis to ensure no differences between control groups were present.
Where data for all the mice are not available, the combined data from the available mice
is presented. For data from the PhIP-ACF rat study, statistical significance was
established by performing 1-way ANOVA, followed by n - 1 custom hypotheses post hoc
tests. Statistical significance was defined as p<0.05.
42
3. Results
3.1 DSS-colitis pilot study
3.3.1. DSS doses of 1.0-3.0% were used to establish a chronic murine model of colitis
with no associated mortality in our animal facility.
All mice in the study survived the entire 9 wk protocol of 3 cycles of 1 wk DSS
water and 2 wk recovery (Figure 2.1). However, the mice exhibited signs of on-going
colonic inflammation such as bloody stools and frank blood on the anus during the DSS-
cycles. Compared to mice in the low-dose 1.0% DSS group, mice treated with higher
doses of DSS had reduced body weight – with a maximal reduction of 22.6 ± 3.2%
(p<0.001) in the 3.0% DSS group (Figure 3.1A), consistent with higher colitis activity.
Moreover, compared to mice supplied with 1.0% DSS in their drinking water, the mice at
higher DSS doses also exhibited marked increases in normalized colon weight, with a
maximal increase of 93.3 ± 13.5% (p<0.001) for the 3.0% DSS group (Figure 3.1B),
suggestive of an increase in fluid infiltration in the colon. As a further indication of
increased disease activity, the colon length (Figure 3.1C) of mice was decreased, to a
maximal of 20.3 ± 2.5% in the 3.0% DSS group, as compared to the 1.0% DSS group (p<
0.001). Moreover, compared to the mice in the 1.0% DSS group, a maximal 88.0 ± 11.8%
(p<0.001) increase in colon weight to length ratio in the 3.0% DSS group was observed,
which was consistent with increased colitis activity.
To perform a histological assessment of disease activity, the colonic damage score
(CDS) was also quantified (Figure 3.2) and was found to be similar for all the
experimental groups in all regions of the colon. Therefore, a dose of 2.5% DSS was
selected for a colitis-associated cancer study (DSS-AOM study) because it did not cause
0.0
10.0
20.0
30.0
1.0% 2.0% 2.5% 3.0%
Bod
y w
eigh
t (g)
0.00
0.01
0.02
1.0% 2.0% 2.5% 3.0%
Col
on w
eigh
t/bo
dy w
eigh
t
0.0
5.0
10.0
1.0% 2.0% 2.5% 3.0%
Col
on le
ngth
(cm
)
* *****
* *** *** ** *** ***
0.00
0.03
0.06
1.0% 2.0% 2.5% 3.0%
Col
on w
eigh
t/le
ngth
(g/
cm)
* *****
Figure 3.1. DSS-colitis pilot study: Body weight, colon colon weight, colon length and colon weight to length ratio. Mice were treated with three cycles of 1 wk DSS (1.0-3.0%, w/v)) and 2 wk recovery to establish chronic colitis and were examined for body weight (A), normalized colon weight (B), colon length (C) and colon weight to colon length ratio (D) as compared to mice treated with 1.0% DSS. n=5-6; *, p<0.05; **, p<0.01; ***, p<0.001 vs. 1% DSS w/v.
A B
C D
43
0.0
4.0
8.0
12.0
16.0
1.0% 2.0% 2.5% 3.0%
CD
S (
Asc
endi
ng c
olon
)
0.0
4.0
8.0
12.0
16.0
1.0% 2.0% 2.5% 3.0%
CD
S (
tran
sver
se c
olon
)
0.0
4.0
8.0
12.0
16.0
1.0% 2.0% 2.5% 3.0%
CD
S (
Des
cend
ing
colo
n)
0.0
4.0
8.0
12.0
16.0
1.0% 2.0% 2.5% 3.0%
Com
posi
te C
DS
Figure 3.2. DSS-colitis pilot study: Colonic damage in mice from Figure 1 was quantified by grading and measuring areas of damage in H&E-stained sections. Colonic damage scores (CDS) in ascending (A), transverse (B), and descending (C) colon were averaged to obtain the composite score (D).
A B
C D
44
45
mortality and was sufficient to induce colitis, with associated weight-losses within the
acceptable limits set by the animal care committee of the University of Toronto.
3.2 DSS-AOM study
3.2.1 Administration of h(Gly2)GLP-2 or antagonism of endogenous GLP-2 with hGLP-
23-33 administration does not alter body weight
Three cohorts of mice (Set 1-3) treated with vehicle control, hGly2GLP-2 or
hGLP-23-33 demonstrated similar trends in body weight-gain throughout the 10-wk
protocol (Figure 3.3). Although a slight decrease in body weight was noted after each
DSS cycle for all three sets of mice (Figure 3.3), mouse body weights on the day of
sacrifice were similar for the vehicle, h(Gly2)GLP-2 or hGLP-23-33 –treated groups
(Figure 3.4).
3.2.2 Administration of hGly2GLP-2 increases small intestinal growth
To determine the functional effects of h(Gly2)GLP-2 and hGLP-23-33
administration, in the three sets of mice, the normalized small intestinal weight, jejunal
villus and crypt lengths and crypt-cell proliferation were examined. For the combined
data from all three sets of mice, administration of h(Gly2)GLP-2 significantly increased
the normalized small intestinal weight by 48.8 ± 5.95% (p<0.001) (Figure 3.5D).
However, blocking the actions of endogenous GLP-2 by administration of hGLP-23-33 did
not change normalized small intestinal weight. Consistent with the change in small
intestinal weight, the jejunal villus and crypt lengths (Figure 3.6A and B) of mice
administered with h(Gly2)GLP-2 were also increased by 42.8 ± 3.5 % and 10.1 ± 2.4%
46
respectively. The administration of hGLP-23-33 did not alter either villus height or crypt
depth in these mice.
Crypt-cell proliferation was also measured by quantifying the number of jejunal
crypt cells positive for the proliferative marker Ki67 on positions 1-20 of jejunal crypts
(Figure 3.6C-D). While all the mice had high proliferation rates at positions 5-15,
administration of h(Gly2)GLP-2 increased proliferation at positions 2 and 20 in the
jejunal crypts (p<0.05-0.01). Notably, administration of hGLP-23-33 reduced proliferation
in positions 10, 14, 16, 17 and 19 (p<0.01-0.01). Together, these results indicate that both
the h(Gly2)GLP-2 and hGLP-23-33 were biologically active in the DSS-AOM mice.
0.0
10.0
20.0
30.0
0 20 40 60 80
Bod
y w
eigh
t (g)
Time (d)
Bicarb
hGLP-2(3-33)
0.0
10.0
20.0
30.0
0 20 40 60 80
Bod
y w
eigh
t (g)
Time (d)
Bicarb
h(Gly2)GLP-2
hGLP-2(3-33)
0.0
10.0
20.0
30.0
0 20 40 60 80
Bod
y w
eigh
t (g)
Bicarb
h(Gly2)GLP-2
Figure 3.3. DSS-AOM study: Body weights. To establish a model of chronic colitis associated with cancer, mice were injected with 10 mg/kg AOM (i.p) and subjected to three cycles of 1 wk DSS in their drinking water (2.5% w/v) followed by 2 wk recovery. The mice were injected with vehicle (Bicarb; Set1-3), hGly2GLP-2 (1.5 μg; Set 1 and 2) or hGLP-23-33(30 ng; Set 1 and 3). The weekly body weight gain for mice in Set 1 (A, n=8-10), Set 2 (B, n=18-19) and Set 3 (C, n=9-26) are as presented. The bars indicate body weight before and after each DSS cycle.
A B
C
47
0.0
10.0
20.0
30.0
Bod
y w
eigh
t (g)
0.0
10.0
20.0
30.0
Bicarb h(Gly2)GLP-2
Bod
y w
eigh
t (g)
0.0
10.0
20.0
30.0
Bicarb hGLP-2(3-33)
Bod
y w
eigh
t (g)
0.0
10.0
20.0
30.0
Bod
y w
eigh
t (g)
A B
C D
Figure 3.4. DSS-AOM study: Body weights on day of sacrifice. The final body weights of mice in Figure 3, Set 1 (A, n=8-10), Set 2 (B, n=18-19) and Set 3 (C, n=9-26) are presented separately. As the body weights on day of sacrifice were similar, this information was combined to demonstrate the body weight on day of sacrifice for all three sets of mice (D, n=27-35).
48
0.000
0.025
0.050
SI
wei
ght/
body
wei
ght
0.000
0.025
0.050
Bicarb h(Gly2)GLP-2
SI
wei
ght/
body
wei
ght
0.000
0.025
0.050
Bicarb GLP-2(3-33)
SI
wei
ght/
body
wei
ght
0.000
0.025
0.050
SI
wei
ght/
body
wei
ght
***
******
*
Figure 3.5. DSS-AOM study: Small intestinal weights. Mice from Figure 3 were examined for their normalized small intestinal weights; Set 1 (A, n=8-10), Set 2 (B, n=18-19) and Set 3 (C, n=9-26) are presented separately. As the normalized small intestinal weights in the corresponding groups were similar, this data was combined in D (n=27-35). *, p<0.05, ***, p<0.001 vs. Bicarb
A B
C D
49
Figure 3.6. DSS-AOM study: Jejunal crypt-villus lengths and proliferation. Jejunalsections of mice from Figure 3 were H&E-stained to measure villus height and crypt depth. Representative H&E-stained jejunal sections (n=10-19) (A) and measurement of villus height and crypt depth (B). Staining for the proliferative marker Ki67 was also performed and representative figures for the positional analysis are presented in (C). The proliferation levels for the jejunum (n=10-24) (D) are as presented. *, p<0.05; **, p<0.01; ***, p<0.001, hGly2GLP-2 vs. bicarb and #, p<0.05; ##, p<0.01; ###, p<0.001, hGLP-23-33 vs. bicarbby1-way ANOVA.
A B
C
Bicarb hGly2GLP-2 hGLP-23-33
50
Bicarb hGLP-23-33
hGly2GLP-2
D
0
25
50
75
100
0 5 10 15 20
Ki6
7 p
osit
ive
cell
s (%
)
Bicarbh(Gly2)GLP-2hGLP-2(3-33)
**
*
##
##
# #
#
-100
0
100
200
300
400
500
600 ***
**
‐
51
3.2.3 GLP-2 does not increase colon growth, but alters chronic colitis damage
To determine whether treatment with exogenous hGly2GLP-2 or blocking
endogenous GLP-2 with hGLP-23-33 administration alter colon growth or inflammation,
the normalized colon weight, length, crypt-cell proliferation and colonic damage score
were quantified. Control mice from all three sets had similar colon weight normalized to
body weight (Figure 3.7). While control mice from Set 1 had colon length similar to
those in Sets 2 and 3, the colon lengths of mice in Set 3 was increased by 13.8 ± 3.84%,
as compared to mice in Set 2 (p<0.01). However, there was no change in colon length
with hGly2GLP-2 or hGLP-23-33 administration (Figure 3.8). Furthermore, analysis of
colonic crypt-cell proliferation revealed that while hGLP-23-33 did not reduce colonic
crypt cell proliferation, administration of hGly2GLP-2 increased proliferation at positions
14, 19 and 20 (p<0.05-0.01) (Figure 3.9A and B). Finally, quantification of colonic
damage by measurement of CDS revealed while hGly2GLP-2 treatment did not alter the
CDS, blocking endogenous GLP-2 action with hGLP-23-33 reduced CDS by 37.3±7.8% (p
< 0.05) (Figure 3.9B). Collectively, these data indicate that while the experimental
treatments altered small intestinal growth, they exerted little to not effect on parameters
of colonic growth.
0
0.01
0.02
Col
on w
eigh
t/bo
dy w
eigh
t
0.00
0.01
0.02
Bicarb h(Gly2)GLP-2
Col
on w
eigh
t/bo
dy w
eigh
t
0.00
0.01
0.02
Bicarb hGLP-2(3-33)
Col
on w
eigh
t/ b
ody
wei
ght
0.000
0.010
0.020C
olon
wei
ght/
body
wei
ght
A B
C D
Figure 3.7. DSS-AOM study: Colon weights. Mice from Figure 3 were examined for their normalized colon weights. Set 1 (A, n=8-10), Set 2 (B, n=18-19) and Set 3 (C, n=9-26) are presented separately. As the normalized colon weights in the corresponding groups were similar, this data was combined in D (n=27-35).
52
0.0
2.5
5.0
7.5
10.0
Col
on le
ngth
(cm
)
0
2.5
5
7.5
10
Bicarb h(Gly2)GLP-2
Col
on le
ngth
(cm
)
0.0
2.5
5.0
7.5
10.0
Bicarb hGLP-2(3-33)
Col
on le
ngth
(cm
)
0.0
2.5
5.0
7.5
10.0C
olon
leng
th (
cm)
Figure 3.8. DSS-AOM study: Colon lengths. Mice from Figure 3 were examined for their and the colon lengths. Set 1 (A, n=8-10), Set 2 (B, n=18-19) and Set 3 (C, n=9-26) are presented separately. As the colon lengths in the corresponding groups were similar, this data was combined in D (n=27-35).
A B
C D
53
0
2
4C
DS
(A
scen
din
g co
lon
)
Figure 3.9: DSS-AOM study: Colonic damage score and proliferation. The colonic damage score was obtained by measuring areas of graded damage in H&E-stained sections of the ascending (proximal) colon of mice in a blinded fashion (A) (n=8-9) and colonic crypt cell proliferation was performed by positional analysis of Ki67 positive cells (B). #, p <0.05 Bicarb vs. hGLP-23-33 and *, p < 0.05; **, p < 0.01 for Bicarb vs. hGly2GLP-2 by 1-way ANOVA
0
25
50
75
0 5 10 15 20
% K
i67
posi
tive
cells
Crypt cell position
Bicarbh(Gly2)GLP-2hGLP-2(3-33)
*
** *
A B
54
#
55
3.2.4 Administration of hGly2GLP-2 and hGLP-23-33 may alter high grade dysplasia and
colon cancer incidence
A gastrointestinal pathologist quantified the incidence of no dysplasia, low- or
high-grade dysplasia (Figure 3.10A-D) and colon cancer (Figure 3.10E-G) in H&E and
AE1/AE3-stained sections of colonic tissue with suspected tumour lesions in a blinded
fashion. It was found that compared to mice treated with vehicle, there was a trend
towards increased high-grade dysplasia and cancer incidence in mice administered with
hGly2GLP-2 (Figure 3.10D,G). Interestingly, the mice treated with vehicle bicarb and
hGLP-23-33 had similar incidences of high- grade dysplasia. However, mice treated with
hGLP-23-33 appeared to have a 28.7% decrease in incidence of colon cancer, while
hGly2GLP-2 treatment increased it by 10% (Figure 3.10G).
3.2.5 All DSS-AOM tumours have high levels of β-catenin; administration of hGly2GLP-2
or hGLP-23-33 does not alter the number of colonic DCAMKL-1-positive stem cells
Immunohistochemistry of tumours slides for the canonical Wnt signaling marker
β-catenin revealed that, while β-catenin was present mostly at the cellular membrane in
normal tissue, it was present in the cytosolic and nuclear fractions in tumour and
dysplastic tissues. Moreover, it was observed that the intensity of β-catenin in tissues
from hGly2GLP-2-treated mice appeared to be greater than in bicarb-treated mice.
The number of DCAMKL-1-positive cells per unit area of normal, dysplastic or
tumour tissue was quantified (Figure 3.11). While the numbers of DCAMKL-1-positive
cells were similar in normal and dysplastic tissue, there were few DCAMKL-1 positive
cells in the tumour tissue. Moreover, compared to the tumour tissue in vehicle bicarb-
D
Figure 3.10. DSS-AOM study: Colonic dysplasia and cancer. A gastrointestinal pathologist examined H&E-stained sections of suspected colonic tissue tissue and identified sections as not dyplastic (A), low-grade dysplastic (B) and high grade dysplastic (C); representative images. The incidence of dysplasia was quantified (D). Tumour tissues stained with AE1/AE3 for epithelial cytokeratins were also examined for tumour invasion and budding for determing cancer incidence. Representative images of normal (E) and tumour (F) tissue and cancer incidence (G) are presented, (n=26-35)
A B C
E
F
G
56
0
10
20
30
40
Bicarb h(Gly2)GLP-2 hGLP-2(3-33)
Can
cer
inci
denc
e (%
)
0
10
20
30
40
50
60
70
No Dysplasia Low-gradedysplasia
High-gradedysplasia
Inci
denc
e (%
)
Bicarb
h(Gly2)GLP-2
hGLP-2(3-33)
0
10
20
30
Normal Dysplasia Tumour
DC
AM
KL
-1 p
osit
ive
cell
s/ m
m2
Bicarb
h(Gly2)GLP-2
hGLP-2(3-33)
Figure 3.11. DSS-AOM study: DCAMKL-1 stem cells and β-catenin expression. Colonic sections from mice with suspected tumour tissues were stained for the intestinal stem cell marker DCAMKL-1 (representative image, A) and the number of cells per unit area of normal, dysplastic and tumour tissue was quantified (B). (preliminary analysis, n=5-7). IHC for the canonical Wnt signaling marker β-catenin was performed, and representative images of normal and dysplastic tissues are presented (C).
A B
57
Nor
mal
Tum
our/
dysp
last
ic
Bicarb hGly2GLP-2 hGLP-23-33C
58
treated mice, there was a trend towards a 72.7 ± 9.8% reduction of these stem cells in the
tumour tissue of mice administered with hGLP-23-33.
3.3 PhIP-ACF study
3.3.1 Feeding AIN-93G diet with PhIP reduces rat body weight
Rats fed with AIN-93G diet mixed with the dietary carcinogen PhIP had slightly
reduced body weight (18.3 ± 1.57%, p<0.01) at the end of PhIP feeding (at 2 wk), as
compared to the rats fed with regular AIN-93G diet (Figure 3.12). After discontinuing the
PhIP diet, all the animals were fed with HF diet and injected with vehicle (Bicarb),
hGly2GLP-2 or hGLP-23-33. Although all the animals gained weight with the HF diet, on
the day of sacrifice, rats in the PhIP+Bicarb group still had a slightly decreased body
weight (10.5 ± 2.07%, p<0.01) as compared to the NoPhIP+Bicarb group (Figure 3.12).
However, amongst the PhIP-fed animals, administration of either hGly2GLP-2 or hGLP-
23-33 did not alter body weight.
3.3.2 Administration of hGly2GLP-2 increases small intestinal but not colon growth
Importantly, while the body weight of animals in the NoPhIP+Bicarb group was
greater than the animals in the PhIP+Bicarb group, the normalized small intestinal and
colon weight and crypt and villus lengths of both groups were not different, indicating
that PhIP feeding per se did not have an effect on intestinal growth (Figure 3.13). To
determine whether hGly2GLP-2 and hGLP-23-33 had functional effects, small intestinal
weight and crypt and villus lengths were measured. It was found that administration of
exogenous hGly2GLP-2 led to a 67.4 ± 4.5% increase (p<0.001) in normalized small
intestinal weight (Figure 3.13A). Moreover, consistent with this increase in small
0
100
200
300
0 10 20 30 40 50
Bod
y w
eigh
t (g)
Time (days)
NoPhIP+Bicarb
PhIP+Bicarb
PhIP+h(Gly2)GLP-2
PhIP+hGLP-2(3-33)
##
Figure 3.12. PhIP-ACF study: Body weight of rats fed with AIN-93G diet with or without 400 ppm PhIP (2 wk) followed by 4 wk of high fat diet with injections of vehicle (Bicarb), hGly2GLP-2 or hGLP-23-33. ##, p < 0.01 vs. NoPhIP+Bicarb
PhIP or NoPhIP+AIN-93G
59
0.00
0.02
0.04S
I w
eigh
t/bo
dy w
eigh
t
NoPhIP PhIP
***
-200
300
800
1300
1800
Cry
pt (
um)
V
illu
s (u
m)
NoPhIP PhIP
***
Figure 3.13. PhIP-ACF study: Small intestinal and colon growth. The normalized small intestinal weight (A), jejunal crypt and and villi (representative images, B), villus height and crypt depth (C), colonic weight (B), and jejunal crypts and villi (representative images, C) and normalized colonic weight of rats from Figure 12 are presented (D). ***, p<0.001 vs. PhIP+Bicarb
0.0000
0.0025
0.0050
0.0075C
olon
wei
ght/
body
wei
ght
NoPhIP PhIP
A B
DC
60
NoPhIP+Bicarb PhIP+Bicarb
PhIP+hGly2GLP-2 PhIP+hGLP-23-33
61
intestinal weight, treatment with hGly2GLP-2 also increased villus height by 43.0 ± 6.3%
(p<0.001) but did not alter crypt depth (Figure 3.13B and C). Notably, hGLP-23-33 did not
elicit any changes in either normalized small intestinal weight or crypt and villus length.
The normalized colon weight was also quantified as a measure of colon growth, but
although there was a small trend of increased colon weight with hGly2GLP-2 treatment,
this change was not significant. Moreover administration of hGLP-23-33 also did not alter
colon weight (Figure 3.13D).
3.3.3 hGly2GLP-2 administration increases the number of ACF in the colon
To determine the effect of hGly2GLP-2 on the development of preneoplastic ACF,
whole colons were stained with methylene blue (Figure 3.14A). Although a few ACF
were detected in the NoPhIP+Bicarb rats, rats in the PhIP+Bicarb group had a 470
±106% increase in the number of ACF, as compared to the NoPhIP+Bicarb group
(p<0.01) (Figure 3.14B). Moreover, PhIP-fed rats treated with h(Gly2)GLP-2
demonstrated an even greater increase (by 72 ± 11%), compared to rats in the
PhIP+Bicarb group (p<0.01). However, while hGLP-23-33-administered rats demonstrated
a trend towards decreased ACF occurrence (by 26 ± 19%); this change was not
statistically significant.
Since larger ACF are associated with greater cancer risk, the occurrence of 1-,2-
or 3-crypt ACF was also quantified. It was found that while administration of hGly2GLP-
2 increased the number of smaller 1-crypt ACF (p<0.05), treatment with hGLP-23-33 did
not have any effect on ACF size (Figure 3.14C). Moreover, the ACF distribution in the
proximal, middle and distal colon was also quantified and found to not differ between the
62
PhIP-fed rats treated with either vehicle bicarb, hGly2GLP-2 or hGLP-23-33 (Figure
3.14D).
3.3.4 GLP-2 does not alter MDF occurrence
The occurrence of MDF was quantified in colons stained with high-iron diamine alcian
blue staining. As for ACF, a small number of MDF were detected in NoPhIP+Bicarb rats
(0.50 ± 0.3 per colon, Figure 3.15). Moreover, the number of MDF found in PhIP+Bicarb
rats was not significantly different from the NoPhIP+Bicarb rats (Figure 3.15).
Furthermore, the number of MDF found in the hGly2GLP-2 and hGLP-23-33-administered
rats were similar to the PhIP+Bicarb-treated rats.
0
5
10
15
Bicarb Bicarb h(Gly2)GLP-2 hGLP-2(3-33)
AC
F co
unt /
col
on
##
**
Figure 3.14. PhIP-ACF study: ACF occurrence. Whole colons of rats from Figure 12 were stained with methylene blue to identify ACF. Representative images of 1-, 2-, 3-crypt ACF are shown in (A). The numbers of ACF by in whole colon (B) and and distributed by size (C) and location within the colon (D) are presented. #, p<0.05; ##, p<0.01 for NoPhIP+Bicarb vs. PhIP+Bicarb and **, p<0.01 for PhIP+Bicarb vs. PhIP+hGly2GLP-2
A
B
C D
0.0
2.5
5.0
Proximal Middle Distal
AC
F co
unt
NoPhIP+BicarbPhIP+BicarbPhIP+h(Gly2)GLP-2PhIP+hGLP-2(3-33)
0
5
10
1 crypt 2 crypt 3 crypt
AC
F co
unt /
col
on
#
*
##
1-crypt ACF 2-crypt ACF 3-crypt ACF2-crypt ACF 3-crypt ACF1-crypt ACF
63
NoPhIP PhIP
0.0
2.0
4.0
Bicarb Bicarb h(Gly2)GLP-2 hGLP-2(3-33)
MD
F c
ount
/ co
lon
NoPhIP PhIP
A
B
Figure 3.15. PhIP-ACF study: MDF occurrence. Whole colons of rats from Figure 12 were stained with high-iron diamine alcian blue stain to identify crypts lacking in mucinstaining. Representative images of normal tissue and MDF (inset) (A) and quantification (B) are presented.
64
65
3.4 PhIP-tumour study
3.4.1 Administration of hGly2GLP-2 does not alter total amount of PhIP consumed or
body weight in rats
The total amount of PhIP eaten by rats for all three groups during the three PhIP-
feeding cycles was not different (Figure 3.16A). Furthermore, administration of
hGly2GLP-2 during early (wk 3-6) or late (wk 15-18) periods did not alter the body
weight of the rats during or after the GLP-2 treatment (Figure 3.16B).
3.4.2 Administration of hGly2GLP-2 during an early phase of cancer development may
lead to increased intestinal tumour burden
During the course of this 60 wk study, 6 rats were found dead or euthanized due
to significant weight loss or other morbidity. Among these, at necropsy, three were found
with intestinal tumours (1 from each group). Moreover, among the remaining animals,
one rat died due to large cell lymphocytic leukemia (Bicarb group), one due to a bleeding
tumour on the head (late hGly2GLP-2) and one due to excessive bleeding from mouth and
anus (early hGly2GLP-2 group). Among animals that completed the 60 wk, the rats given
the early (wk 3-6) treatment with hGly2GLP-2 appeared to have an increased incidence of
suspected intestinal tumours (77.8 %) as compared to rats injected with vehicle control
(42.8%) (Figure 3.17). However, rats treated given the late (wk 15-18) treatment appear
to have a reduced (25%) tumour burden (Figure 3.17). A pathological analysis of these
tissues is currently in progress.
0
350
700
Bicarb h(Gly2)GLP-2 (early) h(Gly2)GLP-2 (late)
Tota
l PhI
P ea
ten/
cage
(mg)
A
B
Figure 3.16. PhIP-tumour study: Total PhIP fed and body weight of rats in the PhIP-tumour study. The rats were subjected to three cycles of PhIP (2 wk) and HF diet (4 wk) followed by 42 wk HF diet. The rats in early GLP-2 group were administered with GLP-2 in wk 3-6 and rats in late GLP-2 group were injected with GLP-2 in wk 15-18. As the rats were doubly-housed, the total PhIP eaten per cage is presented in (A) and the body weights of rats throughout the study is presented in (B). (n=9-10)
66
0
100
200
300
400
500
600
0 13 26 39 52 65
Bod
y w
eigh
t (g
)
Time (week)
Bicarb
h(Gly2)GLP-2 (early)
h(Gly2)GLP-2 (late)PhIP
PhIP
PhIP
hGly2GLP-2 (early)
hGly2GLP-2 (late)
0
50
100
Bicarb h(Gly2)GLP-2 (early) h(Gly2)GLP-2 (late)
Inte
stin
al c
ance
r in
cide
nce
(%)
Figure 3.17. PhIP-tumour study: Occurrence of suspected intestinal tumours in from Figure 17. (Preliminary data, pathological analysis in progress, n=7-9)
67
68
4. Discussion
GLP-2 is an intestinotrophic peptide that is secreted by the enteroendocrine L-cell
in response to nutrient ingestion [213]. GLP-2 has a number of beneficial actions in the
gut, including increased mucosal growth, blood flow and digestive and absorptive
function [2; 7; 8; 55]. Moreover, treatment with GLP-2 effectively reduces the severity of
inflammatory damage and improves mucosal integrity in acute models of DSS colitis and
TNBS ileitis in mice [10; 11; 59]. Hence, the long-acting GLP-2 analog teduglutide, is
now in clinical trials for the treatment of Crohn’s disease [102] and is pending Food and
Drug Administration approval for the treatment of short bowel syndrome. However,
considering that the main action of GLP-2 in the gut is increased mucosal growth, it is
essential to study the potential carcinogenicity of such long-acting GLP-2 analogs.
Moreover, it is well known that patients with colitis-associated IBD (e.g. Crohn’s colitis
and UC) are at increased risk of developing colon cancer and, hence, blocking the action
of endogenous GLP-2 might be an attractive chemo-preventative approach [12; 103;
209]. Since previous reports examining a role for GLP-2 in murine carcinogenesis have
been contradictory, this thesis examined the role of GLP-2 in colon carcinogenesis of a
murine colitis-associated CRC model and a rat model of diet-induced CRC [205; 206;
209].
In this thesis, I demonstrate that, in the DSS-AOM model of murine CRC
associated with colitis, treatment with the exogenous GLP-2 analog hGly2GLP-2 appears
to slightly increase, while blocking endogenous GLP-2 decreases cancer incidence. This
novel finding suggests that the growth-promoting effect of GLP-2 may outweigh its anti-
inflammatory effect in CRC associated with colitis. To determine a DSS dose to be used
69
for the current study, a pilot experiment suggested that compared to the lowest
concentration tested (1.0%, w/v), the higher DSS concentrations (2.0-3.0%, w/v) also
increased the surrogate measures of disease activity – reduction in body weight, increased
colon weight and colon weight to length ratio – without significant morbidity. However,
there were no differences in the CDS, a measure of architectural colitis damage [11; 210].
Since none of these DSS concentrations was linked to mortality or excessive body weight
reduction, a medium 2.5% concentration was selected for the DSS-AOM cancer study.
In the DSS-AOM study, the effects of hGly2GLP-2 and hGLP-23-33 on intestinal
growth were examined mainly in the small intestine, because the jejunum is known to be
the primary target of the mucosal growth action of GLP-2 [2]. Consistent with previous
findings in models of inflammation and in healthy mice, treatment with hGly2GLP-2
increased small intestinal weight as well as jejunal crypt and villus length and crypt cell
proliferation [2; 10; 11; 51; 209]. However, unlike the findings of Iakoubov et al, who
used the same dose of hGLP-23-33 but for 4 wk instead of 2 wk, treatment with 30ng
hGLP-23-33 did not reduce small intestinal weight, suggesting that the duration of
treatment was not adequate [209]. Nonetheless, the finding of reduced small intestinal
crypt cell proliferation in these mice confirmed a functional effect of hGLP-23-33
treatment. Moreover, unlike the effects of hGly2GLP-2 and hGLP-23-33 in the healthy gut,
neither peptide altered normalized colon weight in this model [11; 209]. This finding was
consistent with the absence of any effect of GLP-2 on colon weight in some, but not all
studies of acute DSS-induced colitis in mice [10; 11]. A possible explanation for these
discrepant results is that in our model of chronic colitis, the peptides did not alter colitis
disease activity and, hence, did not alter colon weight and length – both surrogate
70
measures of colitis disease activity. Nonetheless, consistent with positional analyses of
crypt cell proliferation in acute models of DSS-colitis, treatment with GLP-2 increased
colonic crypt cell proliferation [10; 11]. This finding contradicts that of Sigalet et al., who
found a reduction in crypt proliferation index in an acute DSS-colitis model, suggesting
either a differential effect of GLP-2 in an acute vs. a chronic colitis setting, or a
discrepancy in proliferation results due to a difference in methodology [59].
Past reports of the anti-inflammatory effects of GLP-2 have examined GLP-2
action in acute models of murine colitis or ileitis with GLP-2 treatment that was either
concurrent or immediately subsequent to DSS- or TNBS-treatment [10; 11; 59]. While
the effect of GLP-2 in an IL-10-/- mouse model of chronic colitis has been studied, the
effect of GLP-2 treatment has not been examined in chronic colitis models with sporadic
periods of disease activity. Although, the DSS-AOM model is primarily a model of CRC
associated with colitis, it can also be considered a model of chronic colitis due to the
induction of repeated intermittent periods of inflammation associated with bloody stools
and weight loss [73]. Indeed, in general, the mice in the present study demonstrated
occult fecal blood (data not shown), as well as weight loss during DSS treatment.
Furthermore, having undergone 2 cycles of DSS+recovery and an additional DSS
treatment, the mice were presumed to be experiencing chronic colitis by wk 9 of the
study. Nonetheless, it was found that when the long-acting GLP-2 analog, hGly2GLP-2
was administered during wks 9 and 10 of the experimental protocol, the CDS, a measure
of structural damage due to inflammation, was similar to that of vehicle-treated mice.
Moreover, treatment with the GLP-2 antagonist hGLP-23-33 significantly reduced the
CDS, suggesting that, chronic colitis could be alleviated by blocking endogenous GLP-2
71
at that late juncture of disease progression. However, a greater dose or duration of
exogenous GLP-2 treatment may have proven beneficial in this model, since it is known
that the benefits of exogenous GLP-2 administration in humans with chronic CD are
dose-specific [102]. Moreover, it is important to consider that CDS is only an indirect
measure of active inflammation, since it does not involve measurement of inflammatory
cytokines or, at least in my study, assessment by a qualified pathologist.
In the DSS-AOM model of murine CRC, AOM is used in conjunction with DSS
to promote CRC by causing colonic inflammation, leading to low-grade dysplasia and
then to high-grade dysplasia and, subsequently, to invasive carcinoma [187]. This model
of CRC induction is distinct from the stochastic model involving formation of
preneoplastic lesions such as ACF and MDF leading to benign adenomatous polyp
formation, which may become invasive carcinoma [187]. Hence, the incidence of
dysplasia, as a possible mechanistic explanation for the increased cancer incidence in
hGly2GLP-2 administered mice was quantified. Interestingly, the incidence of high-grade
dysplasia in the hGly2GLP-2-treated mice trended towards an increase, as compared to
the vehicle-treated mice. This finding suggests a possible role for exogenous GLP-2 in
the “progression” stage of CRC, from low- to high-grade dysplasia and cancer. It also
adds to the current knowledge that exogenous and endogenous GLP-2 have a role in
increasing preneoplastic lesion formation in the AOM murine model of sporadic CRC
[209]. Additionally, this increase in high-grade dysplasia observed with hGly2GLP-2
treatment is also consistent with the finding of increased ACF formation in PhIP-fed rats,
that is, in another species and with another carcinogen. However, in the PhIP-ACF study,
blocking endogenous GLP-2 action with hGLP-23-33 did not significantly alter ACF
72
incidence, suggesting that either the dose of hGLP-23-33 was inadequate or that
endogenous GLP-2 does not play a role in rat CRC.
Finally, blocking endogenous GLP-2 with hGLP-23-33 lead to a trend towards
decreased cancer incidence in a murine model of colitis-associated cancer. Moreover,
preliminary results indicate that, consistent with increase in ACF in hGly2GLP-2 treated
rats, there is an increase in intestinal tumour incidence in rats treated with hGly2GLP-2 in
the early stages of colon carcinogenesis. Thus, my experiments with the murine DSS-
AOM model, as well as with the rat ACF and tumour induction protocols, have, for the
first time, demonstrated a potential role for exogenous and endogenous GLP-2 in
inducing colonic dysplasia and possibly tumour genesis in two different species.
Previous studies have demonstrated a role for intestinal crypt stem cells as the
cells driving not only normal intestinal growth and repair, but also tumorigenesis [214;
215; 216; 217; 218; 219]. There are known to be two distinct stem cell niches within the
intestine, the crypt-base columnar cells that lie at the base of the crypt and cells that lie
further upward on the crypt, at approximately position 4 [217; 220]. Clevers et al
demonstrated that the stem cells at the crypt base could be effectively identified by
expression of the orphan GPCR, leucine-rich repeat-containing G protein-coupled
receptor 5 (Lgr5) [214]. Moreover, the Lgr5 cells have been demonstrated to be rapidly-
cycling cells that can induce intestinal cancer and are upregulated in other models of
CRC [214; 217; 221]. However, the difficulty of identifying these cells lies in the absence
of reliable antibodies that cross react with Lgr5. In contrast to the rapidly-cycling Lgr5
stem cells at the crypt base, the stem cells at the +4 position are quiescent [217].
Moreover, it has been demonstrated that the +4 cells, that can be identified by the
73
presence of doublecortin and calmodulin-kinase like-1 (DCAMKL-1), are proliferative in
both normal animals and in the adenomas of ApcMin/+ mice [216]. In the DSS-AOM mice,
the effect of exogenous and endogenous GLP-2 on the number of these DCAMKL-1
positive stem cells was therefore examined. Consistent with the cancer incidence data,
there was a trend towards decreased stem cell numbers in tumour tissues with
endogenous GLP-2 blockade using hGLP-23-33 treatment. Interestingly, the number of
DCAMKL-1 stem cells was similar in normal and dysplastic tissues of vehicle,
hGly2GLP-2 and hGLP-23-33-administered mice, suggesting a differential effect of GLP-2
on stem cells, depending on the microenvironment. However, analysis of other types of
stem and progenitor cells is required to assess the complete effect of GLP-2 on cancer
stem cell growth. Thus, collectively, the findings of the DSS-AOM study demonstrate
that while, exogenous hGly2GLP-2 may increase cancer incidence by driving high-grade
dysplasia to tumorigenesis, endogenous GLP-2 may modify cancer growth through
induction of stem cell growth, in murine CRC associated with colitis.
Critique/Limitations. Although the results of my study are intriguing, there are a
number of limitations that must be noted:
1) Sample sizes and statistical analyses: For the DSS-AOM study, the experiments were
performed with three cohorts of mice to a final n value of 26-35 per treatment. As the
colonic proliferative indices have not yet been assessed for all the mice, the results have
been presented with statistical analyses of the currently available data. Additionally,
cancer incidence did not reach statistical significance, although the findings were
indicated as showing a trend, needing further proof by increasing the sample size.
Similarly, for the PhIP-tumour study, the results concerning intestinal tumour occurrence
74
are preliminary and have yet to be confirmed and classified by a gastrointestinal
pathologist. I acknowledge that the final results and conclusions of these studies may
change when the full data set becomes available.
2) Pathological analyses for inflammation: In both the DSS-colitis pilot and DSS-AOM
study, CDS was used as the only measure of inflammation and was not analyzed by a
pathologist, as conventionally performed. Moreover, further analysis of active
inflammation such as pro/anti-inflammatory cytokine quantifications and macrophage
infiltration should be conducted in future studies.
3) The relevance of rodent models to the human condition: The rodent models of
carcinogenesis in these studies were chosen for their similarities to human CRC.
Similarly, the DSS-AOM model of murine CRC was chosen for its ability to link colon
carcinogenesis with the added risk factor of chronic inflammation – the same mechanism
through which human IBD is linked to colon cancer development. However, a major
limitation of this model is the highly accelerated phase of CRC development and high
efficiency of carcinoma development. In humans, it requires years to accumulate the
genetic mutations necessary for cancer development, because of genetic variability and
environmental factors [12]. Moreover, unlike induced inflammation in the mouse model,
humans have inflammatory “flares” at sporadic intervals that may be separated by years
or decades. In addition, the phenotype of the DSS-AOM model is also known to vary
between different strains of mice and depending on the facility [204]. However, the
genetic mutations and molecular pathways involved in CRC development of the DSS-
75
AOM model are similar to those found in human beings [204]. Similarly, for the rat
studies with PhIP, this common dietary carcinogen was used, in combination with HF
diet containing the transfat-rich vegetable oil Primex. However, the concentration of PhIP
used in this study (400 ppm) far exceeds that found in normal human diets [161].
Moreover, it is found that the exposure to low doses, such as those found in the human
diet, may not have carcinogenic potential [222]. However, the mutations caused by PhIP
in the rodent models (e.g. in APC and β-catenin) are also found in human
adenocarcinomas [170; 171; 223]. Hence, although both the murine DSS-AOM and rat
PhIP-HF diet models have points of similarity with the human condition, the relevance of
findings from these studies should be understood in context of the vast differences that lie
between an experimental rodent models and human CRC.
4) The effect of GLP-2 at the initiation/post-initiation or progression phase of
carcinogenesis: The DSS-AOM study was designed to study the effect of GLP-2 on colon
cancer incidence in an inflammation-associated colon cancer model. However, to study
this in more detail, the effect of GLP-2 on both the initiation phase of CRC development
and in the post-initiation phase should have been examined. In my DSS-AOM studies,
GLP-2 treatments were given during the last phase of recovery, e.g. wk 9-10 of the
experimental protocol. As this time period mostly likely corresponds to the progression
phase of carcinogenesis in this model, it remains to be seen whether GLP-2 alters colon
cancer incidence when injected during periods of active inflammation, which would more
closely reflect the initiation phase of carcinogenesis. Similarly, for the PhIP-ACF and
PhIP-tumour studies, it is difficult to assess whether GLP-2 affects the initiation or
76
progression of CRC in this model. Although an increase in the numbers of preneoplastic
lesions such as ACF with hGly2GLP-2 treatment suggests that GLP-2 increases cancer
initiation, ACF cannot be considered as true measures of carcinogenesis, as they do not
demonstrate a number of features of neoplasia and do not correlate with synchronous
adenoma presence [224]. Moreover, with regards to the treatment of early vs. late period
GLP-2 treatments, it was assumed that the early treatment targeted cancer initiation,
while the late treatment was post-initiation/progression. However, these assumptions may
not be true since the timeline of CRC progression in this model is not known.
Consequently, these studies did not directly examine the effect of GLP-2 on initiation or
progression of CRC in the rat-PhIP model.
In summary, the results of my studies indicate that the degradation-resistant GLP-2
analog hGly2GLP-2 may have a slight promoting effect on pre-neoplastic lesions and
cancer development in rodent CRC models. In the murine DSS-AOM model, blocking
endogenous GLP-2 with hGLP-23-33, for 2 wk in DSS-AOM mice and 4 wk in PhIP-fed
rats, was well tolerated, with no discernible effects on small or large bowel weight.
However, it is remarkable that the treatment with hGLP-23-33 appeared to reduce cancer
incidence in a model of colitis-associated cancer. The mechanism underlying this change
occurs could involve effects of GLP-2 on stem cells. The results of these studies may be
relevant to patients being treated with long-acting GLP-2 analogs, in that they may
require more vigilant screening for possible CRC development. Moreover, the cancer-
reducing effects of hGLP-23-33 in the DSS-AOM model need further elucidation for its
development as a possible chemopreventive agent.
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