Aspirin, Non-Steroidal Anti-Inflammatory Drugs, Cyclooxygenase Inhibition and Cancer

Aspirin, non-steroidal anti-inflammatory drugs and cancer

Joe Connor BSc Cellular & Molecular Medicine

Introduction Word Count: 2829 Total Word Count: 18772

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Abbreviations 15-LOX-1 – 15-Lipoxygenase-1 15-PGDH – 15-Prostaglandin De-hydrogenase 5-FU – 5-Flurouracil 6CEPN – 6-C-(E-phenylethenyl)-naringenin AA – Arachidonic Acid ACF – Aberrant Crypt Foci AOM – Azoxymethane APC – Adenomatous Polyposis Coli APC Trial – Adenoma Prevention with Celecoxib Trial APPROVe Trial - Adenomatous Polyp Prevention on Vioxx Trial Atg - Autophagy Genes bFGF – Basic Fibroblast Growth Factor BaP – Benzo(a)pyrene c-Met – Hepatocyte Growth Factor CAC – Colitis Associated Cancer cAMP – Cyclic Adenosine Monophosphate CaPP – The Cancer Prevention Project CDDO-Me – 2-cyano-3,12-dioxooleana-1,9(11)-dien-C28-methyl ester CDK – Cyclin-dependent Kinase CIMP – CpG Island Methylator Phenotype CIN – Chromosomal Instability CK1 – Casein Kinase 1 COX – Cyclooxygenase DMH – Dimethylhydrazine ECM – Extra-cellular Matric EGF – Epidermal Growth Factor EGFR – Epidermal Growth Factor Receptor EIF4e-BP1 – Eukaryotic Translation Initiation Factor 4E-Binding Protein EP – Prostaglandin E Receptor FAP – Familial Adenomatous Polyposis GSK3β – Glycogen Synthase Kinase 3 β HAA – Heterocyclic Aromatic Amines hCOX-1 – Recombinant Human Cyclooxygenase 1 HIF-1 – Hypoxia-Inducible Factor 1 HNPCC – Hereditary Non-Polyposis Colorectal Cancer HR – Hazard Ratio HRE – HIF Responsive Element IGF – Insulin Growth Factor IL-1α – Interleukin 1 α LGR5 – Leucine-rich Repeat-containing G-protein Coupled Receptor 5 MDA – Malondialdehyde MDR – Multi-drug Resistance Protein MMP – Matrix Metalloproteinase

3 MMR – Mismatch Repair MRP4 – Multi-drug Resistance Protein 4 MSI – Microsatellite Instability mTOR – Mammalian Target of Rapamycin NO – Nitric-oxide NO-aspirin – Nitric-oxide Releasing Aspirin NSAIDs – Non Steroidal Anti-Inflammatory Drugs PGD2 – Prostaglandin D2 PGE2 – Prostaglandin E2 PGEM – Prostaglandin E2 Metabolite PGES – Prostaglandin E Synthase PGF2α – Prostaglandin F2α PGI2 – Prostaglandin I2 PGH2 – Prostaglandin H2 PGP – P-glycoprotein PHD – HIF Prolyl Hydroxylase PHS – The Physicians’ Health Study PI3K – Phosphatidylinositol-3-kinase PKA – Protein Kinase A PKB – Protein Kinase B (Also referred to as Akt) PreSAP Trial – Prevention of Sporadic Adenomatous Polyps Trial RR – Relative Risk SNP – Single Nucleotide Polymorphism TGF-β – Transforming Growth Factor β TNF-α – Tumour Necrosis Factor α TXA2 – Thromboxane A2 uPAR – Urokinase Plasminogen Activator Receptor VEGF – Vascular Endothelial Growth Factor VHL – Von Hippel Lindau WHS – The Women’s Health Study

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Table of Contents 1. Introduction ........................................................................................................................................ 7 1.1 Colorectal Cancer .......................................................................................................................... 7 1.2 Linking Anti-Inflammatory Activity to Reduced Cancer Risk....................................................... 13 1.3 Observing Chemoprevention: Epidemiological Studies and Clinical Trials ................................ 15 1.4 Evaluation of Targets for Cancer Chemoprevention: Determining the Tumourigenic Functions of the COX Enzymes. ......................................................................................................................... 18 1.5 Aims of This Dissertation............................................................................................................. 19 2. Observing Chemoprevention: Epidemiological Studies and Clinical Trials Using Aspirin and Other NSAIDs .................................................................................................................................................. 21 2.1 The Effect of Aspirin on Sporadic Cases of Colorectal Cancer .................................................... 21 2.2 The Effect of Aspirin on Recurrence of Disease .......................................................................... 27 2.3 The Effect of Aspirin on the Prevention of Colorectal Cancer in High Risk Groups .................... 30 2.4 The Effect of Other NSAIDs on Colorectal Polyps in High Risk Groups ....................................... 33 2.5 The Effect of Other (COX-2 selective) NSAIDs on Colorectal Polyps in Lower Risk Groups ........ 36 3. COX-2 in Tumourigenesis ................................................................................................................. 39 3.1 Introduction to COX-2/PGE2 Signalling ....................................................................................... 39 3.2 Tumourigenic Functions of COX-2/PGE2 Signalling – Increased Replicative Potential ............... 42 3.3 Tumourigenic Functions of COX-2/PGE2 Signalling – Decreased Sensitivity to Anti-growth Signals ............................................................................................................................................... 46 3.4 Tumourigenic Functions of COX-2/PGE2 Signalling – Suppression of Apoptosis ........................ 48 3.5 Tumourigenic Functions of COX-2/PGE2 Signalling – Sustained Angiogenesis ........................... 51 3.6 Tumourigenic Functions of COX-2/PGE2 Signalling – Increased Metastatic Potential................ 53 3.7 Tumourigenic Functions of COX-2/PGE2 Signalling – Adaptation to the Tumour Microenvironment ............................................................................................................................ 56 3.8 Tumourigenic Functions of COX-2/PGE2 Signalling – Maintenance of Stemness ....................... 62 3.9 Tumourigenic Functions of COX-2/PGE2 Signalling – Resistance to Chemotherapy................... 64 3.10 The Role of COX-2 in Tumourigenesis - Concluding Statements .............................................. 66 4. COX-1 in Tumourigenesis ................................................................................................................. 67 4.1 Early Evidence for COX-1 Involvement in Tumourigenesis ......................................................... 67 4.2 Elucidating the Role of COX-1 in Tumourigenesis – Evidence for Early Involvement ................. 69 4.3 Elucidating the Role of COX-1 in Tumourigenesis – Activation of Carcinogens ......................... 70

5 4.4 Elucidating the Role of COX-1 in Tumourigenesis – Lessons from the Protection of Intestinal Stem Cells .......................................................................................................................................... 73 4.5 Elucidating the role of COX-1 in Tumourigenesis – First Suggestions of a Mechanism for COX-1 Mediated Tumourigenesis ................................................................................................................ 74 4.6 Lessons from Investigations into COX-1 – the Significance of 15-PGDH in Colorectal Cancer ... 78 4.7 Lessons from Investigations into COX-1 – Novel Strategies Exploiting the Role of 15-PGDH in Tumourigenesis ................................................................................................................................. 78 4.8 Directions for Further Study – Developing Novel COX-1 Inhibitors ............................................ 80 4.9 The Role of COX-1 in Tumourigenesis – Concluding Statements................................................ 81 5. Concluding Statements .................................................................................................................... 82

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Abstract BACKGROUND: Colorectal cancer is one of the major causes of morbidity and mortality in developed nations. Incidence remains high due to poor uptake of screening procedures and is increasing in some nations. There is a significant requirement for effective primary preventative strategies.

AIMS: Numerous questions remain over the use of non-steroidal anti-inflammatory drugs (NSAIDs) in the chemoprevention of colorectal cancer. Here, the adequate dosage and period is characterised and the complex roles played by both the cyclooxygenase (COX) isoforms COX-2 and COX-1, the targets of NSAIDs, in colorectal tumourigenesis are described. Components of these pathways may act to determine colorectal cancer risk and NSAID response.

FINDINGS: Low dose aspirin, around 75mg per day, can protect against colorectal cancer, albeit after a delay. Short term benefits may be derived from clearance of pre-malignant cells. However, significant gastrointestinal and cardiovascular toxicity is associated with COX-1 and COX-2 inhibition respectively. COX-2 is thought to contribute to tumourigenesis at a later stage and additional roles in adaptation to the tumour microenvironment and maintenance of cancer stem cells may imply therapeutic benefit from its inhibition. COX-1 putatively plays an earlier role, with the accumulation of basal PGE2 levels possibly contributing to tumourigenesis downstream of 15-PGDH loss. These findings may allow for personalised allocation to NSAID prophylaxis, depending on the inherent benefit of the patient.

CONCLUSIONS: NSAIDs clearly prevent colorectal cancer through their inhibition of the COX enzymes. Perturbations in the COX-mediated mechanisms of tumourigenesis can be used to drive personalised direction to NSAID prophylaxis.

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1. Introduction 1.1 Colorectal Cancer Cancer is one of the major causes of morbidity and mortality in the world, with an estimated 14 million new cases in 2012 (1). Colorectal cancer made up 9.1% of these cases and was responsible for 8.4% of the 8.2 million cancer-associated deaths (1).

Industrial nations display the highest rates of colorectal cancer incidence for both men and women (see Figure 1), with diet and lifestyle having been shown in recent decades to be crucial risk factors for cancer generally (2, 3) and particularly colorectal cancer (4). However, in recent years some regions with traditionally low rates of colorectal cancer incidence are beginning to see an increase (see Figure 2), thought to be due to an increasingly ‘Westernised’ lifestyle (5) and the inability of poorly established medical infrastructures to implement preventative strategies, such as faecaloccult-blood screening, sigmoidoscopy and colonoscopy. These ‘secondary’ preventative strategies are currently endorsed by medical professionals and their effect on the incidence of colorectal cancer is clear (6). However, though clearly effective in most settings, they can be invasive and it has recently been demonstrated that even in countries with good medical infrastructures, the number of individuals actually undergoing these procedures is low (7). Hence there is a significant need for effective primary preventative strategies.

Primary Prevention involves the implementation of strategies in a low or average risk population that prevent the onset of a disease. These strategies often use drugs to interfere with the pathogenesis of a disease, such as the adenomacarcinoma sequence of colorectal cancer, preventing an individual from developing even sub-clinical disease (8), where patients are asymptomatic but disease progression has begun.

Secondary Prevention refers to the prevention of clinically manifested disease in those who have already developed sub-clinical disease (9). In terms of cancer, this could be the removal of either colorectal polyps by exploratory colonoscopy or the entire colon by colectomy, should the risk of disease be high enough.

Box 1 – The difference between primary and secondary prevention

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Figure 1 – The estimated age-standardised incidence rates of colorectal cancer worldwide per 100,000 men (left) and women (right) in 2012 Darker blue indicates a higher level of incidence and it can be seen from these graphs that colorectal cancer is typically a disease of more developed nations, often thought to be due to lifestyle and environmental factors.

Image from Ferlay et al (1)

Cancer arises due to both hereditary and somatic genetic aberrations, lifestyle, exposure to carcinogens, diet and chronic inflammatory states (3, 10, 11). Colorectal cancer can arise through multiple pathways. The common sequence of colorectal cancer carcinogenesis sees an early mutation in a gene that controls cellular proliferation, such as the tumour suppressor gene adenomatous polyposis coli (APC). Inactivation of the second APC allele through further mutation results in the loss of heterozygosity required to initiate the generation of an adenoma. A subset of adenomas may undergo further mutations and epigenetic events in crucial growth control genes become more malignant, generating a carcinoma (see Figure 3) (12). This common pathway of tumourigenesis is also associated with major chromosomal translocations or deletions, as such it is referred to as the chromosomal instability pathway (CIN) (13).

This mechanism of increasing malignancy with distinct morphological changes is commonly referred to as the adenoma-carcinoma sequence (see Figure 3).

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Figure 2 – The trends in the incidence rate of colorectal cancer in nations with classically high and low rates of colorectal cancer, per 100,000 men (top) and women (bottom) These data show that nations with classically low levels of colorectal cancer, such as those in the Far East and less developed nations in Europe are beginning to see significant increases in recent years. This is thought to be due to numerous factors including adoption of a more ‘Western’ lifestyle. Trends such as these highlight the need for further preventative strategies.

Image from Ferlay et al (1)

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Another common pathway in colorectal tumourigenesis is the microsatellite instability pathway Figure 3 – The adenoma-carcinoma sequence characteristic of the development of colorectal cancers.

Though colorectal cancer can arise through distinct pathways, the adenoma-carcinoma sequence is characteristic of colorectal tumourigenesis in both sporadic and hereditary cases. The accumulation of oncogenic mutations or epigenetic changes in important proto-oncogenes, such as KRAS, or tumour suppressor genes, such as APC or TP53, drives the development of carcinomas. In pre-disposition syndromes, such as Familial Adenomatous Polyposis (FAP) and Hereditary Non-Polyposis Colorectal Cancer Syndrome (HNPCC), individuals inherit one mutated allele of the APC gene in the former and in genes that control the process of mis-match repair (MMR) in the latter, which pre-disposes HNPCC patients to a ‘mutator phenotype’, in which a number of genes may become mutated. As such, these individuals are more likely to develop adenomas.

Image from Davies et al (14), with modifications.

Another common pathway in colorectal tumourigenesis is the microsatellite instability pathway (MSI). This mechanism for colorectal cancer development is responsible for 15% of sporadic cases and more than 95% of cases of hereditary non-polyposis colorectal cancer syndrome (HNPCC) (15). It is characterised by inherited mutations in crucial DNA repair genes involved in the process of mismatch repair (MMR) (16). It has been shown that mutation in MMR genes can lead to a 100-fold increase in the mutation rate in the colorectal mucosa (16) and that these germline mutations convey a significantly increased risk of developing colorectal cancer (17).

The CpG island methylator phenotype (CIMP) has been proposed as another distinct pathway of colorectal tumourigenesis, though this is challenged by some. This pathway is characterised by aberrant methylation and inactivation of a wide range of genes, including crucial growth control genes, such as INK4A (p16) and MLH1, involved in MMR (18, 19), though there remains debate as to exactly which genes are most affected in this pathway (20). CIMP is thought to contribute to overlap

11 between pathways and may be responsible for a subset of sporadic MSI cancers. Furthermore, recent studies have highlighted the fact that a CIMP phenotype may be conducive to the acquisition of oncogenic mutations (21), which themselves may contribute to enhancing the CIMP phenotype (22). Studies are continuing to further define the CIMP phenotype and its interaction with other recognised pathways of colorectal tumourigenesis.

Around 3-5% of colorectal cancers arise due to hereditary predisposition syndromes such as Familial Adenomatous Polyposis (FAP), characterised by the inheritance of a mutated APC allele and Lynch Sydrome (HNPCC), in which a mutated allele of various MMR genes is inherited (23). With both the more common sporadic and hereditary cases of colorectal tumourigenesis unified by the adenomacarcinoma sequence of tumourigenesis, FAP and HNPCC patients provide useful models to study colorectal pathogenesis.

Chronic inflammatory conditions of the bowel, such as ulcerative colitis and Crohn’s disease, have been identified as risk factors for colorectal cancer. The risk of developing colorectal cancer increases with time in inflammatory bowel disease (IBD) patients (24). The severity of inflammation may be indicative of colorectal cancer risk, with several epidemiological studies suggesting that the histological level of inflammation is associated with an increased likelihood of colorectal cancer onset (25, 26), suggesting a causal relationship.

The inflammatory mechanism of colorectal cancer carcinogenesis, often referred to as colitis associated cancer (CAC), has been putatively identified as a distinct mechanism of colorectal tumourigenesis (27) and is thought to share numerous mechanisms with previously identified pathways involved in cases of colorectal cancer that do not arise from an inflammatory background (see Figure 4).

It is worth mentioning that CAC and the classically defined pathways of colorectal tumourigenesis may not be mutually exclusive. Since regular use of aspirin, an anti-inflammatory drug, was

12 associated with a decreased incidence of colorectal cancer in HNPCC patients (28), it may be the case that mediators of inflammation plays a key role in the other pathways of colorectal tumourigenesis.

A significant body of evidence suggests that cancer is a highly preventable disease, estimated in a A Figure 4 – The similarity between mediators of tumourigenesis in both the CAC (blue) and classically defined (CIN, MSI, CIMP, yellow) mechanisms of colorectal cancer development The two mechanisms are thought to share many of the same mediating events, such as increased activity of Kras and loss of P53. Interestingly, some similarities are thought to have been derived from different mechanisms. It has been hypothesised that Ă&#x;-catenin may become aberrantly active in CAC through mechanisms independent of APC mutation (29), which is detected only infrequently in CAC carcinomas (30, 31).

Both mechanisms, however, have events specific to eachother. CAC is often stimulated by constitutively increased activity of pro-inflammatory cytokines and their receptors, while spontaneous colorectal cancer is often stimulated by loss of the key tumour suppressor APC. ACF = Aberrant Crypt Foci Image from Terzick et al, with alterations (27)

A significant body of evidence suggests that cancer is a highly preventable disease, estimated in a landmark study to be around 35% (2). This is specifically relevant for sporadic cases of colorectal cancer, whose risk factors, which include numerous dietary factors, obesity, exercise and chronic inflammation (32), indicate it may be inherently preventable. Indeed, it has been suggested that lifestyle and behaviour does not need to be drastically altered in order to provide a significant

13 reduction in risk, estimated by some to potentially be as high as 70% (33). There is a significant requirement for an enhancement of the primary preventative strategies, due to the increasing incidence of colorectal cancer in developing nations and the stubbornly high levels in developed countries. Furthermore, due to the fact that metastases have already been established in around 20% of patients that present with colorectal cancer (34), decreasing severely their likelihood of survival, prevention of cancer is key.

1.2 Linking Anti-Inflammatory Activity to Reduced Cancer Risk It is clear, therefore, that inflammation plays an important role in colorectal cancer. In addition, inflammation is involved in the aetiology of numerous pathologies, including cancer – discussed previously – and Alzheimer’s disease among others (35). In humans, the lipid-like hormone prostanoid family are significant mediators of inflammation. The cyclooxygenase (COX) enzymes are responsible for their production.

The COX enzymes act through the sequential oxidation and reduction of arachidonic acid (AA), which is liberated from cellular membranes through the action of phospholipases (36). These reactions result in production of prostaglandin H2 (PGH2). This product acts as the pre-cursor molecule for the production of all the prostanoids, which play various roles in numerous tissues, with specific synthase enzymes producing the specific PGH2 derivatives (36) (see Figure 5).

It has been known since the seminal work of J. R. Vane that aspirin and other NSAIDs achieve their anti-inflammatory activity through inhibition of the production of prostaglandins (37). It was later made clear that this activity was mediated through the inhibition of the COX enzymes responsible for their production (38, 39).

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Figure 5 – The prostaglandin cascade The COX enzymes mediate the conversion of AA, liberated from cellular membranes, into the pre-cursor prostaglandin, PGH2. This is then converted by specific tissue isomerases into the prostanoids required in those tissues. For example, prostaglandin E2 (PGE2) is largely produced in the kidneys, the brain and the gastric mucosa, where it plays a protective role. PGE2 acts through binding to G-protein coupled receptors, termed EP receptors, found on the surface of intracellular and extracellular membranes. There are additional products liberated from PGH2, which have also been putatively identified as having roles in the tumourigenesis of various cancers: thromboxane A2 (TXA2) has been shown to have roles in cell proliferation and invasiveness and metastasis (40-42). Prostaglandin F2ι has been shown to potentially have a role in generating a more motile phenotype in colorectal cancer (43), while prostaglandin D2 (PGD2) may in fact have an inhibitory effect on tumourigenesis (44, 45).

Image from Ricciotti et al, with alterations (46)

There are two main COX isoforms: COX-1 and COX-2, though a third has been identified (47). Its role in humans is still unclear and therefore will not be discussed in this dissertation. COX-1 is thought to function as a housekeeping gene. It is constitutively expressed and through a constant level of activity provides a basal level of prostaglandins. These prostaglandins have been implemented in many homeostatic processes, including the maintenance of the lining of the gastric mucosa (48).

15 Indeed, the inhibition of this isoform and its function by aspirin is thought to be responsible for the severe gastric bleeding associated with regular aspirin use (49).

COX-2, however, is not constitutively expressed in most tissues and is upregulated in certain scenarios. The induction of COX-2 expression occurs particularly in response to inflammatory stimuli (50, 51). Inhibition of COX-2 is also associated with severe adverse effects, potentially limiting the clinical feasibility of COX-2 specific NSAIDs. Since various NSAIDs with different specificities have been shown to inhibit the COX enzymes and have been shown to be potentially chemopreventative, a significant body of work now exists to determine the function of the COX enzymes and the COX-derived prostaglandins, particularly prostaglandin E2 (PGE2), in tumourigenesis.

1.3 Observing Chemoprevention: Observational Studies and Clinical Trials Using Aspirin and Other NSAIDs The field of chemoprevention has increased in importance in regard to colorectal cancer specifically, but also many other cancers. The field largely grew in response to various epidemiological and observational studies that coincidentally suggested a link between NSAIDs and a decreased risk of developing or dying from colorectal cancer. ‘Self-selected’ NSAID users, regularly using the drugs to relieve the symptoms of inflammatory conditions such as rheumatoid arthritis, and individuals allocated to aspirin for the prevention of cardiovascular events largely provided the first evidence for NSAID-mediated chemoprevention.

Both aspirin and other NSAIDs have been classically linked to a decreased incidence of colorectal cancer (52, 53) and significant polyp regression in high-risk cohorts (54-56), though uncertainties over dosage and period of prophylaxis cast doubt over whether these drugs, particularly aspirin, had a significant chemopreventative effect (57, 58). The optimisation of these conditions will be discussed in this dissertation.

16 In subsequent decades, work has shifted to further characterise the effects that these drugs may have on the incidence and mortality of colorectal cancer in populations of varying risk of colorectal and other forms of cancer in order to determine more accurately those that would benefit most from NSAID prophylaxis. There is a wealth of data regarding the chemopreventative effects of aspirin derived from cardiovascular protection studies and hence this will largely be the focus of this dissertation. This data has been the subject of numerous meta-analyses and has recently been reviewed (52, 53, 59-62). While taking cancer incidence and mortality data from these studies provides questionable statistical power, the findings have still been valuable. Namely, there is a growing consensus that aspirin has a significant chemopreventative effect in average-risk individuals, albeit after a significant delay of around ten years (59, 63). However, it has also been established that regular use of aspirin is associated with significant adverse effects, including gastrointestinal bleeding and potentially haemorrhagic stroke (64, 65). The benefit-to-hazard ratio that balances the risk of these significant side effects with the potential chemopreventative benefit to an individual will remain a caveat to the findings of the trials discussed throughout this dissertation.

Aspirin and other ‘older’ NSAIDs, such as sulindac, ibuprofen, piroxicam and others, are seen as relatively non selective COX-inhibitors (See Figure 6). Though they have a degree of specificity for a certain COX-isoform, and for simplicity when discussed in detail later they will be separated by their specificities to a particular COX isoform at clinically relevant concentrations, they may, in the case of aspirin and sulindac, inhibit both COX isoforms at sufficient concentrations.

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Figure 6 – NSAIDs have varying specificities for the two COX isoforms Assays such as the William Harvey Human Modified Whole Blood Assay (WHMA) can determine the activity of certain drugs. The IC80 ratio of NSAIDs for COX-2 and COX-1 can determine the selectivity of these drugs for a particular isoform.

Some routinely used NSAIDs, such as rofecoxib and celecoxib are selective for COX-2. Others, such as sulindac (sulindac sulphide) are <5-fold selective for COX-2 and can inhibit both isoforms, while aspirin is slightly more selective for COX-1 but can also inhibit both isoforms at sufficient concentrations.

Image from Warner et al, with modifications (66)

In more recent years with the aim of improving efficacy and decreasing adverse effects, more selective NSAIDs have been developed and have also been suggested to have a chemopreventative effect in both significantly higher (67) and slightly lower risk individuals (68-70). However, these selective NSAIDs were also reported to be causally linked to a number of severe cardiovascular side effects (71, 72), providing a significant obstacle to their clinical feasibility. Therefore, this dissertation will discuss the potential advantages and disadvantages of both non-selective aspirin and selective NSAIDs in the chemoprevention of many cancers, but particularly colorectal. It will emphasise the importance of the hazard-to-benefit ratio for different populations throughout and discuss the research that must be undertaken in the future to more accurately determine the effectiveness and clinical feasibility of NSAID-mediated chemoprevention.

18 1.4 Evaluation of Targets for Cancer Chemoprevention: Determining the Tumourigenic Functions of the COX Enzymes.

Elucidating exactly how these enzymes contribute to colorectal tumourigenesis has been an invaluable tool in determining the clinical feasibility of COX inhibition and for determining modifications within these pathways that stratify groups that may benefit most from NSAID prophylaxis.

Since COX-2 overexpression has been shown to be a common and crucial event in colorectal tumourigenesis (73-75), numerous studies have been undertaken to determine the mechanisms by which it mediates tumourigenesis and characterise the basis for the side effects associated with its inhibition. Increased COX-2 expression has been shown to be capable of inducing tumourigenesis in transgenic mice (76). Through the signalling of its pro-inflammatory product, PGE2, COX-2 has been intrinsically linked to a number of the classical hallmarks of cancer, including roles in mediating enhanced cellular proliferation (77-80), insensitivity to anti-growth signals (81), resistance to apoptosis (81, 82) and roles in enhancing metastasis (83-85). Inhibition of these processes provides the basis for primary chemoprevention. However, recent work has ascribed numerous activities to COX-2 that may provide the basis for additional benefits of COX-2 inhibition over the timeline of cancer development, including interactions with the tumour microenvironment (86-88) and potential maintenance of cancer stem cells (89). These processes will be discussed at length in this dissertation to determine whether COX-2 inhibition may also provide benefit as a secondary chemopreventative or indeed a therapeutic strategy.

While most of the literature regarding NSAID-mediated chemoprevention is focused on the inhibition of COX-2, there is a growing body of evidence to suggest that COX-1 may play a role in colorectal tumourigenesis and hence its inhibition may partly explain the chemopreventative effects attributed to aspirin and other non-selective or COX-1 selective NSAIDs. This dissertation will review the evidence for COX-1 involvement, including early knockout and immunohistochemical staining studies (90, 91), pharmacokinetic analysis of drugs shown to have a chemopreventative effect (92,

19 93) and the first studies that determined pathologically relevant roles for COX-1 in mediating colorectal tumourigenesis (94, 95). Numerous questions remain regarding the feasibility of COX-1 inhibition as a chemopreventative strategy. One of the most important challenges is to define exactly what stage of colorectal tumourigenesis, if any, that is mediated by COX-1 will determine whether its inhibition will prevent cancer onset. Further, with significant toxicity classically associated with regular use of both specific and non-specific NSAIDs, the benefit-to-hazard ratio for independent groups must be determined. This dissertation will review the recent advances regarding both COX-2 and COX-1 and characterise the clinical feasibility of their inhibition.

1.5 Aims of this Dissertation It is well established that prevention of colorectal cancer is an important and potentially achievable goal. Epidemiological and clinical evidence strongly suggests that NSAIDs have chemopreventative properties. However, given the significant toxicity associated with regular NSAID use, this dissertation will aim to determine whether chemoprevention with NSAIDs is a clinically feasible option for all individuals, or whether these strategies should be reserved for specific patients.

It is important to characterise the mechanisms by which these drugs have this effect. Hence, an aim of this dissertation will be to discuss the numerous roles attributed to COX-2 in colorectal tumourigenesis and highlight the growing evidence for and suggested mechanisms played by COX-1 in colorectal tumourigenesis. Since the majority of the work carried out in recent decades has focused on COX-2, these mechanisms will be characterised in depth here. A major focus, however, will be given to more recently suggested mechanisms of COX-2 mediated tumourigenesis, such as the interaction with the tumour microenvironment and potential maintenance of cancer stem cells.

However, this dissertation will also aim to review the evidence for and suggested mechanisms of COX-1 in colorectal tumourigenesis, despite the relative paucity of studies in this area. Though COX-1 has largely been ignored, there is sufficient evidence to suggest that it plays an important role in

20 tumourigenesis and the recent elucidation of a putative model for COX-1 mediated tumourigenesis may have wide ranging clinical applications, including the explanation of NSAID resistance and the identification of new targets and methods of chemoprevention.

Understanding the underlying mechanisms of NSAID-mediated chemoprevention will allow for a more precise stratification of individuals who would benefit from such strategies, potentially allowing for individualised chemoprevention. Most importantly of all, this dissertation will tie together the findings of epidemiological and clinical trials and the various studies that investigate the roles of the COX enzymes in colorectal tumourigenesis to establish the groups that would potentially benefit most from these strategies.

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2. Observing Chemoprevention: Observational Studies and Clinical Trials Using Aspirin and Other NSAIDs Numerous epidemiological studies and clinical trials have been undertaken to determine the activity of various NSAIDs as chemopreventative agents of numerous diseases, including cancer. These provide useful data as to the populations that benefit most from NSAID prophylaxis and allow further characterisation of the adequate dosage and period in order to minimise toxicity. Further, elucidating the effect of NSAIDs in different populations with different risks of colorectal cancer allows clarification of the processes involved in colorectal tumourigenesis and the ways NSAIDs may prevent these processes.

2.1 The Effect of Aspirin on Sporadic Cases of Colorectal Cancer The very earliest evidence for a chemopreventative role for aspirin was discovered coincidentally in observational studies for which prevention of cancer was not the primary endpoint. While this limits the statistical power of these findings, the findings are useful and have paved the way for further, more targeted trials that are currently underway.

Early randomised, double-blinded studies were undertaken in an attempt to more accurately determine this relationship. The first of these was the Physiciansâ&#x20AC;&#x2122; Health Study (PHS) (58). Previous observational studies have involved individuals who had themselves for various reasons chosen to use NSAIDs. Studies involving these individuals are unlikely to provide useful data as the reasons for choosing to use NSAIDs, such as inflammatory conditions such as rheumatoid arthritis may alter the risk of developing certain forms of cancer.

With excellent results coming early in the PHS, which aimed to determine the effect of aspirin on cardiovascular complications and beta carotene on cancer incidence, the aspirin portion of the study was ended early. Hence, the follow-up period for this group was only five years (58).

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Confounding is a statistical occurrence in which the characteristics of the trial cohort may influence the measured end point along with your measured exposure (96). For example, self-selected users of aspirin may have chosen to do so due to a condition that modulates risk of developing colorectal cancer.

Box 2 – The principle of confounding This highlights the potential issue that arises with taking data from early studies, as since prevention of cancer was not the primary endpoint crucial data may be missed. Despite having a five year follow up, results were somewhat encouraging (See Table 1). While daily aspirin did not seem to prevent the incidence of invasive cancer, individuals taking daily aspirin were at a lower risk of developing polyps or cancer in situ (58).

A similar study, the Women’s Health Study (WHS) called the chemopreventative effect of aspirin into question. This study suggested that the women who were allocated to alternate day, low dose aspirin were not protected from developing cancer generally (57). However this, like the PHS, may have suffered from an inappropriate design to properly determine a chemopreventative effect for aspirin. The individuals in this study were taking alternate day 100mg doses of aspirin. Since the target of aspirin is dose dependent and its inhibition of COX-2 is thought to only occur at higher concentrations, it may be the case that the dosage of aspirin used in this study may not have been high enough to provide an accurate measure of chemoprevention. Indeed, further studies have highlighted the requirement for daily dosage for a chemopreventative effect to be seen (63).

Recently, the follow-up period of this study was extended and it was suggested that the group allocated to aspirin did indeed see a decreased risk of developing overt colorectal cancer when data was taken from the extended ‘post-trial’ period compared to the original trial period (97). This latent protection corroborates with other studies that suggest that chemoprevention of new cancers by aspirin requires prophylaxis for a number of years (52, 53, 61).

An analysis of the nutrition cohort of the Cancer Prevention Study II, in which a subset of individuals taking aspirin daily were monitored for cancer incidence, reported that aspirin seemed to decrease

23 Table 1 - Effect of aspirin on the incidence of and/or mortality from sporadic cases of colorectal cancer in average risk individuals Trial The Physicians' Health Study (58)

Study Population 22071 male physicians: 11037 allocated to aspirin, 11034 to placebo

Aspirin Dosage 375mg every other day

Main Findings Incidence of colorectal polyp / cancer in situ: RR = 0.86 (95% CI = 0.68 - 1.10) Incidence of invasive cancer: RR = 1.15 (95% CI = 0.80 - 1.65)

The Women's Health Study (57, 97)

39876 women: 19934 allocated to aspirin, 19942 to placebo

100mg every other day

Incidence of colorectal cancer [original trial period]: RR = 0.97 (95% CI= 0.77 - 1.24) Incidence of colorectal cancer [extended trial period]: HR = 0.58 (95% CI = 0.42 – 0.80)

Cancer Prevention Study II (98)

146113 individuals

‘Adult 325mg≥ daily

Strength'

Incidence of cancer (men, <5 years use): RR = 0.97 (95% CI = 0.91 - 1.03) Incidence of cancer (men, 5> years use): RR = 0.84 (95% CI = 0.76 - 0.93) Incidence of cancer (women, <5 years use): RR = 1.05 (95% CI = 0.99 - 1.11) Incidence of cancer (women, 5> years use): RR = 0.86 (95% CI = 0.73 - 1.03) Incidence of colorectal cancer (both, 5> years use): RR = 0.68 (95% CI = 0.52 - 0.90)

Pooled data from five randomised trials (53)

16488 individuals: 10737 allocated to aspirin, 5751 to placebo

Varying Doses: 75mg – 1200mg daily

Incidence of colorectal cancer (75-300 mg/day aspirin): HR = 0.75 (0.56-0.97) Incidence of colorectal cancer (75-1200mg/day aspirin): HR = 0.76 (0.60 – 0.96) Incidence of colorectal cancer (Aspirin doses ≤300 mg/day, treatment period ≤ 2.5 years) HR = 0.69 (95% CI = 0.51 - 0.93) Incidence of colorectal cancer ( Aspirin doses ≤300 mg/day, treatment period ≥ 5 years) HR = 0.62 (95% CI = 0.43 - 0.94)

Pooled data from eight randomised trials (61)

25570 individuals: 14035 allocated to aspirin, 11535 to placebo

Varying Doses: 75mg - 325mg daily

Risk of death due to colorectal cancer (≤5 years follow-up): HR = 0.78 (95% CI = 0.39 1.56) Risk of death due to colorectal cancer (≥5 years follow-up): HR = 0.41 (95% CI = 0.17 1.00)

Pooled data from 51 randomised trials (62)

77549 individuals: 40269 allocated to aspirin, 37280 to placebo

Varying Doses: 30mg - 325mg daily

Odds of developing cancer (≤3 years follow up): OR = 1.01 (95% CI = 0.88 - 1.15) Odds of developing cancer (≥3 years follow-up): OR = 0.76 (95% CI = 0.66 - 0.88)

HR = Hazard Ratio – The relative rates of a certain event occurring in two groups, one with and one without intervention (for example aspirin and placebo groups) OR = Odds Ratio – A measure of the association between a certain intervention and a pre-determined outcome. A determination of the odds of a certain event occurring with and without intervention.

RR = Relative Risk – A ratio of the risk of an event occurring in one group, for example an aspirin cohort, and the risk in another, such as a placebo group.

24 the risk of developing both cancer generally and specifically colorectal cancer (98). This chemopreventative effect became more marked with increased aspirin dosage time.

Both this study and others that preceded it tended to be observational studies and hence their findings may have suffered from confounding (Box 2). For example, the cohort analysed by Jacobs et al. were â&#x20AC;&#x2DC;self-selectedâ&#x20AC;&#x2122; users. They had inflammatory conditions that required daily aspirin and these conditions may have contributed to differing risks of cancer in these populations. Hence, there remained a significant need for stringently regulated randomised and blinded clinical trials to more definitively define the protective effects of prophylactic aspirin use.

Recently, several meta-analyses of numerous observational and clinical trials have been undertaken

by Rothwell et al. to answer the numerous questions that remain regarding aspirin-mediated chemoprevention (53, 61, 62). These analyses involved trials whose primary endpoint was the prevention of adverse cardiovascular events, such as myocardial infarction and stroke, but also involved the collection of individual patient mortality and cancer incidence data. Through analysing these trials in combination and in many cases extending the follow-up periods of these trials, these meta-analyses have allowed an appreciable characterisation of aspirin mediated-chemoprevention.

In the first of these studies, low dose aspirin, defined as 30-300mg daily, was associated with a substantial decrease in the 20 year risk of developing colorectal cancer (see Table 1) (53).

Furthermore, it was established in this analysis that dosage period appears to have an effect on the protective effects of aspirin, since those allocated to a longer scheduled dosage benefitted significantly from this extended prophylaxis, both in terms of incidence and mortality (53). This analysis also went some way to answer questions regarding the optimum dosage of aspirin required for beneficial chemoprevention. It suggested that those allocated to higher doses of aspirin saw no additional prevention from colorectal cancer; indeed it is suggested that receiving doses as low as 75 mg/day were derived similar benefit to those allocated to much higher doses (53). Since regular use

25 of higher doses of aspirin is associated with significant toxicity, chemoprevention derived from lowdose aspirin would be beneficial to the patient.

Extended follow-ups in a second major meta-analysis by this group also yielded interesting results: it was determined that the majority of the benefit from daily aspirin prophylaxis is only demonstrable after substantial periods of follow-up post-intervention, with at least 5 years thought to be necessary (See Table 1) (61). For some cancers, particularly colorectal, the period of follow-up required to determine benefit appears to be longer, perhaps as long as 10 years post-intervention (61). This may represent the relatively slow carcinogenesis and hence long periods of time required for colorectal cancer to develop.

What still remains unclear, however, is whether significant short-term benefits can be gained from daily prophylactic use of aspirin. In a pooled analysis of individual data from a large number of randomised trials, it has been suggested that a chemopreventative effect can be observed as soon as three years after the trial period had ended (See Table 1) (62). This analysis also provided an updated model on the clinical feasibility of prophylactic aspirin use. It established an evolving model of risk and benefit when stratification by follow-up period was undertaken. After short periods of follow up, protection from vascular events predominates. However, with increasing follow-up, this group suggested that while protection from cancer onset remains, the additional activities of aspirin, namely protection from vascular events and major extra-cranial bleeds, do not continue for extended periods once intervention has ceased (62). It must be mentioned, however, that clinical scenarios involving long-term use of daily aspirin would not follow the model of these studies exactly, since aspirin use was limited to a relatively short period and was scheduled to end.

Interestingly, the findings of this study do not corroborate with previous findings that a significant â&#x20AC;&#x2DC;lagâ&#x20AC;&#x2122; period exists before protection from cancer incidence and mortality is seen (53, 61). This could reflect the still unclear role of aspirin in chemoprevention. An early observed effect on cancer mortality would reflect an activity that perhaps clears already established malignant cells and

26 prevents metastasis, an activity that has been observed in observational studies (60). It was suggested by this group that inhibition of the roles played by platelets in promoting metastatic spread of tumours (99) may be an explanation for the reduced number of cancer deaths in those allocated to aspirin (62). A latent effect would suggest a role in inhibiting the early stages of cancer development, as cancers take many years to form and the effects wouldn’t be seen until this normal timeline has progressed.

A key caveat in the field of aspirin-mediated chemoprevention has been the numerous safety concerns associated with daily use. A number of adverse effects, including extracranial bleeding – most commonly of the gastrointestinal tract – peptic ulcer and haemorrhagic stroke, have been associated with regular aspirin use with higher doses conferring a higher risk of adverse effects (64, 65). A recent review determined the benefit-harm balance of prophylactic aspirin in the major studies that have occurred in this field (59). Using parameters established in previous studies – in which protection required around 5 years of aspirin use and was observable after a delay of around 10 years – Cuzick et al. calculated the overall benefit of aspirin prophylaxis. Through the calculation of the absolute risk reduction of both cancer and cardiovascular complications and the absolute risk increase of major side effects, a slight net benefit was calculated for both men and women (59), though this depended on the characteristics of the individual.

Consequently, direction of individuals towards aspirin and other NSAID prophylaxis will have to be based on the risk of side effects in that individual. Since alcohol consumption and Helicobacter pylori infection have been shown to increase the risk of aspirin-mediated bleeding (100, 101), it is thought that additional variables may also determine the balance of the benefit-to-harm ratio. Furthermore, further elucidation of factors that contribute to NSAID-induced adverse effects, such as genetic factors that may predispose to gastrointestinal bleeding, may be necessary in determining whether aspirin prophylaxis is of benefit on an individual basis.

27 While it is important to determine the effect of NSAIDs on sporadic cases of colorectal cancer in average risk individuals, the natural timeline of cancer onset means that prospective trials would have to be undertaken over an extremely long period, during which time the risk of adverse effects may be too severe. Hence, studying individuals with previous colorectal polyps is an attractive model. These individuals have an inherently increased risk of developing polyps again, allowing for shorter trials to be designed whilst still characterising the effect of NSAIDs. 2.2 The Effect of Aspirin on Recurrence of Disease With analyses of various trials casting doubt on the chemopreventative effect of regular aspirin in low risk groups (57) and more recent meta-analyses of similar trials suggesting such a relationship did exist (53, 61), it is worth noting that some studies have been undertaken to determine whether regular use of aspirin could prevent recurrence of colorectal adenomas in patients whose previously detected adenomas had been removed by colonoscopy. One of these studies showed that allocation to aspirin decreased the incidence of both ‘early’ polyp and ‘late’ polyps, differentiated by various histological characteristics (See Table 2) (102). When taken with other findings that regular use of aspirin upon, but not before, diagnosis of disease is associated with a decreased risk of cancer death (103), it can be suggested there may be a potential role for aspirin to be taken therapeutically upon diagnosis of disease or as a secondary preventative strategy.

However, not all studies in this field suggest a role for aspirin in secondary chemoprevention. A recent analysis disputed the results reported by Logan et al. (102), suggesting that allocation to aspirin after diagnosis and removal of adenomatous polyps had no real effect on the recurrence of polyps

in

a

four-year

follow

up

period

(See

Table

2)

(104).

28 Table 2 - The effect of aspirin on the prevention of recurrence of disease

Trial

Study Population

NSAID + Dosage

ukCAP Trial (102)

853 patients with previous Aspirin - 300mg daily colorectal polyps: 434 allocated to aspirin, 419 allocated to placebo

APACC Trial (104)

185 patients with previous Aspirin - Either 160mg or 1 Year (Interim) Results (105): colorectal polyps: 55 allocated to 300mg daily Risk of developing colorectal adenomas (low dose aspirin): low dose aspirin, 47 to higher dose RR = 0.85 (95% CI = 0.57 – 1.26) aspirin, 83 allocated to placebo Risk of developing colorectal adenomas (higher dose aspirin): RR = 0.61 (95% CI = 0.37 – 0.99) 4 Year Results: of developing colorectal adenomas (both aspirin groups): RR = 0.95 (95% CI = 0.75 1.21) Average size of polyp: Aspirin group = 3.1 ± 5.8mm, placebo group = 3.4 ± 6.2mm

Colorectal Adenoma 517 patients with a previous Aspirin - 325mg daily Prevention Study (106) colorectal adenoma: 259 allocated to aspirin, 258 allocated to placebo

Main Findings Risk of developing colorectal adenomas: RR = 0.79 (95% CI = 0.63 0.99) Risk of developing advanced colorectal adenomas: RR = 0.63 (95% CI = 0.43 - 0.91)

Risk of developing a new colorectal adenoma: RR = 0.64 (95% CI = 0.46 - 0.91) No significant difference between the two groups in the risk of developing advanced adenomas

29 Furthermore, when polyps were detected again, allocation of aspirin seemed to have no effect on the overall polyp burden in these patients, determined by the average size of detected polyps (104).

The contrast between the findings of this group and others led to this group suggesting that aspirin may have varied activities that can be regarded as chemopreventative. With shorter studies (102) and interim analysis by Benamouzig et al. (105) suggesting that aspirin prevents recurrence of polyps, but this longer study finding that no such relationship exists, it has been suggested that when taken in the short term aspirin may display an anti-tumour activity, removing the precancerous polyps, the mechanisms for which are described later. However, it may the case that after this time aspirin shifts its activity to a chemopreventative one; preventing the generation of these polyps.

A similar rationale to using individuals with previous polyps is using FAP and HNPCC patients, since these conditions are characterised by a significantly enhanced risk of developing cancer and go through

similar

mechanisms

to

sporadic

cases.

30 2.3 The Effect of Aspirin on the Prevention of Colorectal Cancer in High-risk Groups Randomized clinical trials using COX-2 selective NSAIDs have highlighted a significant chemopreventative effect with regular use of these drugs in high-risk individuals, discussed later (67, 107). However, such trials had not been undertaken until recently regarding the use of aspirin in these patients. The majority of the data described above regarding aspirin-mediated chemoprevention comes from trials where prevention of cardiovascular events was the primary endpoint. Hence, there has been a significant need for such trials to be undertaken.

The characterisation of such a relationship has been attempted by the Cancer Prevention Project (CaPP) trials. These trials can hence be seen as the first aspirin trials where prevention of precancerous polyps was seen as the primary endpoint. The first of these trials attempted to determine the effect of aspirin on the generation of polyps in young FAP patients. This trial found that those allocated to either aspirin arm were largely unprotected from polyposis, but determined that aspirin had a significant effect on the general size of polyps in these patients (see Table 3) (108). These results provide insight into the activity of aspirin as a chemopreventative drug. A beneficial effect on the size of polyps as opposed to their number would indicate a role in inhibiting the mechanisms associated with the increasing malignancy of these polyps, as opposed to their instigation.

The second CaPP trial involved a similar design, but used Lynch Syndrome patients. Crucially, this was the first trial in the field to use prevention of colorectal cancer as its primary endpoint, rather than the generation of pre-cancerous polyps. In contrast to the findings of CaPP1, this trial found that regular use of aspirin was associated with a significant decrease in the risk of developing colorectal cancer, as well as the many other cancers to which HNPCC predisposes, albeit after a delay (see Table 3) (28). Interestingly, this trial provided evidence that aspirin retains its chemopreventative activity even in the absence of an inherently inflammatory background, further highlighting its potential importance as a chemopreventative agent.

31 While these findings contrast with those of CaPP1, the characteristics of aspirin in this trial are similar to those reported by previous studies, albeit while using higher doses of aspirin, suggesting that use of aspirin must occur for a number of years before protection is detected. It should be noted that the accelerated generation time of cancers in Lynch Syndrome patients means that it is to be expected that the delay period is shorter than that of low-risk individuals (28).

The conflicting results of the first two CaPP trials means that further investigation is required. While ethical questions are naturally raised regarding performing placebo-controlled trials in individuals of such a high risk of cancer, it is important that the questions that remain are answered. Similarly to the studies involving low risk individuals, one of these is the dosage of aspirin required to give a chemopreventative effect. The first two CaPP trials used far higher doses of aspirin than have previously been reported to be effective in average-risk populations (59). Hence, the CaPP3 trial, which began recruiting in October 2014 and is due to be completed by 2021, will attempt to determine the effective dosage of aspirin required to see a potential chemopreventative effect in Lynch Syndrome patients (109).

32

Table 3 - The effect of aspirin on the incidence of polyps in high-risk groups Trial

Study Population

NSAID + Dosage

CaPP1 (108)

133 young FAP patients: 62 Aspirin - 600mg daily allocated to aspirin, 71 allocated to placebo

Main Findings Risk of developing increased numbers of colorectal polyps: RR = 0.77 (95% CI = 0.54 1.10) Overall polyp burden was significantly decreased in the aspirin group

CaPP2 (28)

861 young Lynch syndrome Aspirin - 600mg daily patients: 427 allocated to aspirin, 434 to placebo

Risk of developing colorectal cancer: HR (<2 years allocation to aspirin) = 1.07 (0.47 - 2.41), HR (â&#x2030;Ľ2 years allocation to aspirin) = 0.41 (95% CI = 0.19 - 0.86) Risk of developing other Lynch-predisposition HR = 0.63 (95% CI = 0.34 - 1.19)

CaPP3 (109)

Recruitment began 2014; continuing

October Aspirin - Either 100mg, 300mg or Trial is due to end in 2021 600mg daily

cancers:

33 2.4 The Effect of Other NSAIDs on Colorectal Polyps in High-risk Groups Numerous trials have been undertaken to determine the effect of NSAIDs on high-risk populations.

Early studies involving families suffering from FAP found that regular use of the commonly prescribed NSAID sulindac shrunk many of the numerous polyps that characterise this disease (55, 110). In these patients, those who had a preventative colectomy, a common procedure for FAP patients, but retained their rectums saw the polyps in this area regress (55); while those who had retained their colon also saw regression of not all, but most polyps with regular sulindac use (55). In response to these early findings, studies were later set up to determine the effect of sulindac on the polyps of FAP patients who had not undergone total colectomy, itself a chemopreventative strategy, to more accurately characterise the effect sulindac had (54). This study found that regular sulindac use was associated with significant regression of polyps; both in terms of size and number (see Table 4). Furthermore, upon the cessation of the sulindac treatment period, polyp number and size determined by exploratory colonoscopy began to increase, further highlighting the effect sulindac was having (54). Unlike previous studies total regression of polyps was not found to occur and regression was greater after 6 months than 9, suggesting for the first time that NSAID resistance may occur during regular use (54).

In a follow-up to this study, this group performed a larger and longer analysis to determine the chemopreventative effect of sulindac in FAP patients. Interestingly, while similar results were seen to previous studies (see Table 4), after the 48 month treatment period, sulindac had a less encouraging preventative effect than previously seen (107). This could add further evidence for the argument for NSAID resistance or a disparity between short and longer term use of NSAIDs, as the effect of sulindac â&#x20AC;&#x201C; so clearly seen in shorter studies â&#x20AC;&#x201C; becomes negligible once the treatment period is

increased.

34

Table 4 - The effect of COX-2 selective inhibitors on the incidence of polyps in high-risk (FAP) groups Trial Giardiello et al. 1993 (54)

Study Population NSAID + Dosage 22 FAP patients: 11 allocated to Sulindac - 150mg twice daily sulindac, 11 allocated to placebo control

Main Findings Polyps reduced in number in the sulindac group by 44% Polyps reduced in size in the sulindac group by 35% Total regression was not observed

Giardiello et al. 2002 (107) 41 FAP patients: 21 allocated to Sulindac - Either 75mg or 150mg A smaller percentage of the sulindac group developed sulindac, 20 allocated to placebo twice daily significant adenomas (43% vs 55%) control The difference in polyp number between the two groups was 0.52 (95% CI = -0.29 â&#x20AC;&#x201C; 2.73) Steinbach et al. 2000 (67)

77 FAP patients: 32 allocated to Celecoxib - Either 100mg or After a 6 month treatment period, polyp number low dose celecoxib, 30 allocated 400mg twice daily decrease: 400mg = 28%, 100mg = 11.9%, placebo = to higher dose celecoxib, 15 4.5% allocated to placebo control After a 6 month treatment period, polyp burden decrease: 400mg = 30.7%, 100mg = 14.6%, placebo = 4.9%

35 In addition to sulindac, new generations of specific COX-2 inhibitors were developed and their chemopreventative effect studied, since COX-2 inhibition is clearly linked with a wealth of chemopreventative mechanisms and COX-2 inhibition was thought to be safer than COX-1 inhibition (36). A landmark study using one of these inhibitors, celecoxib, suggested that these inhibitors had a significant effect on the polyps of FAP patients, decreasing both number and overall burden of polyps in a dose-dependent manner (see Table 4) (67). It should be mentioned that since the placebo group also saw a decrease in polyp number and burden, there may have been confounding factors that may have altered the results.

Nonetheless, studies using more COX-2 selective inhibitors have reached a workable consensus regarding their prophylactic use in high risk patients. Rather than replacing traditional secondary chemopreventative strategies such as sub-total or total colectomy, NSAIDs may be utilised as an adjuvant or follow-up therapy, to prevent the further generation of polyps in parts of the colorectum that are left intact.

Crucially, as safety concerns concerning NSAIDs that inhibit both COX isoforms, such as aspirin, were partly the reason for studying highly COX-2 selective inhibitors, celecoxib was reported in this study to be largely safe. However, with various retrospective analyses suggesting that regular use of COX-2 inhibitors is associated with cardiovascular toxicity (71, 72), albeit with some suggesting the celecoxib is the safest (111), it may be the case that relatively short studies such as the one undertaken by Steinbach et al may not have appreciated the potential long-term side effects of regular COX-2 inhibitor use.

Since hereditary pre-disposition syndromes make up a minority of colorectal cancer cases, the effect of NSAIDs must be characterised in average-risk populations to determine their widespread clinical feasibility.

36 2.5 The Effect of Other (COX-2 selective) NSAIDs on Colorectal Polyps in Lower-risk Groups With COX-2 selective inhibitors having been clearly linked to regression of the numerous polyps characteristic of FAP patients, it remained to be determined whether these drugs could be protective against sporadic cases of colorectal adenomas in lower-risk groups.

Such a study, the Prevention of Sporadic Adenomas Prevention (PreSAP) trial, found that regular use of daily celecoxib was associated with a decreased risk of developing both early and advanced colorectal adenomas (see Table 5) (68). Similar results were seen in the Adenoma Prevention with Celecoxib (APC) trial, which saw individuals allocated to varying doses of daily celecoxib (70). It found that, similar to previous studies, long-term regular use of this COX-2 inhibitor was associated with a reduction in risk of developing both early and especially advanced adenomas, in a dose-dependent manner (see Table 5) (70). While the preventative effects were similar to previous studies, this trial suggested a significantly higher risk of adverse side effects than previous studies. This study suggested that those allocated to celecoxib may be three times as likely to experience an adverse cardiovascular event, such as myocardial infarction, than those allocated to placebo (70). Such a significant risk of adverse effects provides a major caveat to the potential use of these drugs as a widespread chemopreventative strategy.

A similar trial, the Adenomatous Polyp Prevention on Vioxx (APPROVe) trial, reported on the effect of regular use of the COX-2 inhibitor rofecoxib in individuals with at least one established adenoma (69). Early results from this trial suggested an encouraging chemopreventative effect (see Table 5) (69). However, the APPROVe trial drew attention to the severity of the side effects associated with regular COX-2 selective NSAID prophylaxis. The risk of cardiovascular events was such that this trial was shut down before its completion, with participants allowed to be un-blinded and their risk evaluated (112). Indeed, this group also reported a significantly increased risk of gastrointestinal complications, not previously reported in COX-2 inhibitor trials, and an increased risk of death in the rofecoxib group (69). The safety concerns associated with rofecoxib, marketed as Vioxx, are such

37 that in 2004 the Medicines and Healthcare Products Regulatory Agency (MHRA) recommended that patients stop taking regular rofecoxib (113).

Therefore, specific COX-2 inhibitors are clearly associated with a decreased risk of developing colorectal adenomas. Specifically, numerous trials report that the greatest protection occurs in regard to the generation of advanced adenomas, perhaps highlighting the role of COX-2 in tumourigenesis, overexpression of which is thought to be a later process in adenoma development (91). However, the commonly reported side effects in each of the trials described above remain a concerning caveat to these encouraging findings. The reporting of these side effects has been the source of controversy in recent years, with some retrospective analyses suggesting that the adverse effects associated with use of COX-2 selective NSAIDs had not been adequately analysed and reported (71, 114). In a similar analysis to the studies regarding aspirin, the utility of COX-2 inhibitors must be balanced against the potential hazards to an individual on a per-patient basis. The significantly increased risk of cardiovascular events with daily COX-2 inhibitor use will mean that some individuals at higher inherent risk of cardiovascular disease may not benefit from the potential chemopreventative effects.

With both COX-1 selective and COX-2 selective NSAIDs having been shown to inhibit both polyp formation and cancer onset, there is a concerted effort to characterise the functions of these enzymes in tumourigenesis.

38

Table 5 - The effect of COX-2 selective inhibitors on the incidence of polyps in lower-risk groups Trial PreSAP trial (68)

Study Population NSAID + Dosage 1397 individuals: 840 allocated to Celecoxib - 400mg daily celecoxib, 557 allocated to placebo

APC trial (70)

2035 individuals: 685 allocated to Celecoxib - Either 200mg or Risk of developing colorectal adenomas: lower dose celecoxib, 671 allocated 400mg twice daily RR (200mg group) = 0.67 (95% CI = 0.59 - 0.77), to higher dose celecoxib, 679 RR (400mg group) = 0.55 (95% CI = 0.48 - 0.64) allocated to placebo Risk of developing advanced colorectal adenomas: RR (200mg group) = 0.43 (95% CI = 0.31 - 0.61), RR (400mg group) = 0.34 (95% CI = 0.24 - 0.50)

APPROVe trial (69) 2376 individuals: 1158 allocated to Rofecoxib - 25mg daily rofecoxib, 1218 allocated to placebo

Main Findings Risk of developing colorectal adenomas: RR = 0.64 (95% CI = 0.56 - 0.75) Risk of developing advanced adenomas: RR = 0.49 (95% CI = 0.33 - 0.73)

(Interim Results) Risk of developing colorectal adenomas: RR (higher risk groups) = 0.76 (95% CI = 0.69 - 0.83), RR (lower risk groups) = 0.76 (95% CI = 0.69 - 0.83) Risk of developing advanced colorectal adenomas: RR (higher risk groups) = 0.72 (95% CI = 0.58 - 0.89), RR (lower risk groups) = 0.70 (95% CI = 0.58 - 0.86) The trial was ended early due to safety concerns

39

3. COX-2 in Tumourigenesis NSAIDs have long been known to exert their activities largely through the inhibition of the COX enzymes. Overexpression of COX-2 is an important event in colorectal tumourigenesis. Therefore, investigating the tumourigenic functions of this isoform allows for a better understanding of the mechanisms of colorectal cancer development and characterisation of how aspirin and other NSAIDs carry out their tumourigenic effects.

3.1 Introduction to COX-2 and PGE2 Signalling The COX enzymes catalyse the first two steps of the synthesis of the pro-inflammatory hormone-like prostanoids (36). While the majority of the prostanoid lipids have been implicated in tumourigenesis (40-45, 115), most of the literature is focused on the role of COX-2 derived PGE2.

COX-2 overexpression is a common event in colorectal tumourigenesis. It is thought that oncogenic mutations, for example in the Wnt and ras signalling pathways, may co-operate to mediate this overexpression (116).

Increased COX-2 expression can occur in numerous cell types in the tumour environment, including the malignant cells themselves (117), local macrophages (118) and stromal myofibroblasts (119), among others. This suggests that COX-2 derived PGE2 signalling can occur at the membranes of cancerous cells by both an autocrine and a paracrine mechanism (see Figure 7).

40

Figure 7 – Overexpression of COX-2 and associated PGE2 signalling occurs by numerous methods A model of both autocrine and paracrine transport and activity of PGE 2 can be concluded from numerous studies. Enhanced COX-2 expression in cancer cells leads to enhanced levels of intracellular and secreted PGE 2 (117). This PGE2 can act in both an autocrine manner, binding to EP receptors on the surface of the cancer cell itself, or by a paracrine mechanism on surrounding stromal or endothelial cells, stimulating further tumourigenic processes, such as angiogenesis (117). Furthermore, cells in the microenvironment can also experience increased COX-2 expression (118) and the PGE2 produced can be secreted and act in both an autocrine and a paracrine fashion. The paracrine activation epithelial cells can mediate a ‘landscaping’ effect, which can directly contribute to tumourigenesis through, for example, the induction of angiogenesis (117).

The COX-2 protein is primarily located and functions at the endoplasmic reticulum and around the nuclear envelope (120), the former of which is also the primary location of PGE2 synthesis (121).

PGE2 predominantly functions through the interaction with membrane-bound receptors, termed EP 1-4 (46). Therefore, in order to stimulate these receptors, PGE2 must be secreted by cells. While this was originally thought to be merely though passive diffusion, a growing number of specific transporters have been elucidated. One such transporter associated with PGE2 efflux from the cell is multidrug resistance protein 4 (MRP4) (122). This transporter has accrued a great deal of interest, as PGE2 must largely be trafficked out of the cell in order to interact with its membrane-bound receptors. Importantly, MRP4 is up-regulated in colorectal cancer cell lines (123), suggesting that increased PGE2 efflux may play a role in colorectal tumourigenesis. A relatively small body of work has shown an inhibition of MRP4 by NSAIDs, such as ibuprofen (124). Though this is an interesting

41 finding, it is questionable how important inhibition of PGE2 transport is in the mechanisms of NSAIDmediated chemoprevention if synthesis of PGE2 itself is blocked through inhibition of the COX enzymes. It has recently been shown that another transporter, involved in the influx of PGE2 from the extracellular milieu that does not allow for the signalling seen during interaction with EP receptors (125), but in fact couples to its degradation (126) and whose importance will be described later, is down-regulated in colorectal cancer (126).

There is a great body of evidence to support the role of the COX-2/PGE2 pathway in colorectal tumourigenesis. COX-2, which is not expressed in normal tissue in the colorectum (127), has been found to be overexpressed in carcinoma tissue, but, as to be expected, not in surrounding tissue (74). This over-expression implies that COX-2 is directly associated to tumourigenesis. Indeed, both mRNA detection and immunohistochemical staining have suggested that COX-2 overexpression occurs in 70-80% of colorectal cancers (75, 128). Various studies to be discussed later have shown that COX-2 overexpression contributes to tumourigenesis by inducing increased levels of PGE2 and its associated signalling pathways. Hence, there is significant interest in the role aspirin and other NSAIDs with COX-2 inhibitory activity may play in the inhibition of these pathways through the inhibition of PGE2 synthesis.

This correlation between COX-2 overexpression and tumourigenesis has been observed in animal models. Landmark studies involving APCÎ&#x201D;716 mice, which are pre-disposed to developing intestinal adenomas and serve as an animal model for FAP, found that both inhibition and knockout of COX-2 significantly reduced the number of polyps they developed (90, 129).

Numerous studies have been undertaken to determine how COX-2 contributes to tumourigenesis, in order to understand how colorectal cancer develops and characterise how NSAIDs have their chemopreventative activities, through its inhibition.

42 3.2 Tumourigenic Functions of COX-2/PGE2 Signalling â&#x20AC;&#x201C; Increased Replicative Potential The acquired ability to proliferate beyond normal cellular constraints is one of the key processes that occurs during tumourigenesis (130). Since COX-2 is thought to be partly responsible for such characteristics at a relatively early stage in tumourigenesis, understanding these processes provides insight into how they can be manipulated in cancer chemoprevention.

The majority of the work regarding the pathways that regulate the increased ability of a cancer cell to proliferate has focused on the Wnt signalling pathway (see Figure 8). This is due to two major findings. Firstly, the Wnt signalling pathway is active at the base of colonic crypts, suggesting a role for Wnt signalling in the preservation of stem cells in the colonic crypt and additional roles as epithelial cells become more differentiated (131). Second, there is a gradient of Wnt signalling, a marker of which was Ă&#x;-catenin activity, throughout the colonic crypt, with the highest activity occurring at the base of the crypt. As cells become more differentiated higher up the crypt, Wnt signalling decreases (132) (See Figure 9). Indeed, knocking out components of the Wnt signalling pathway results in loss of the intestinal crypts (133).

These findings highlighted the role of Wnt signalling in the homeostasis of the intestinal epithelium and hence contributed to the belief that this pathway, which can be activated by PGE2 in mechanisms discussed below, is deregulated in colorectal cancer. This hypothesis is supported by studies that show that mutations within this pathway are intrinsically associated with colorectal cancer. Mutations in the APC gene, a key controller of Ă&#x;-catenin activity during Wnt signalling (134), are found in 80% of sporadic colorectal cancers (135). Furthermore, hereditary conditions that involve the inheritance of a mutant APC gene, such as FAP, confer an almost guaranteed risk of developing colorectal cancer (135).

43

Figure 8 – The canonical Wnt signalling pathway – showing both the classically accepted mechanism (A) and the more recently suggested mechanism (B) The Wnt signalling pathway is a key process during the growth processes involved in embryogenesis and in the maintenance of many adult tissues (134). Its deregulation is thought to be an early event in colorectal tumourigenesis. Numerous studies have shown that COX-2 derived PGE2 can positively regulate this crucial growth pathway through mechanisms discussed later.

Image from Clevers et al (136)

Figure 9 – The gradient of Wnt signalling in healthy and diseased intestinal crypts In healthy intestinal crypts, there is a gradient of Wnt signalling from the base to the top of the crypt. As the crypt becomes diseased, and the APC gene is lost, this gradient is disrupted and the cells in the crypt exhibit significant dysplasia. Image from Boman et al (137), with alterations

44

A growing body of work has linked the COX-2/PGE2 signalling pathway with activation of this key proliferative signalling pathway. Through its interaction with its G protein coupled receptor, EP2, PGE2 has been shown to activate cyclic AMP (cAMP) (138). Through this activation, PGE2 activates Protein Kinase A (PKA). PKA is involved in two key regulatory phosphorylation events that act to mediate Wnt signalling (77, 78). PKA phosphorylates GSK3ß in an inhibitory fashion on Serine 9 (78).

Since this protein provides the key priming phosphorylation of ß-catenin in its degradation in the absence of Wnt stimulation (136), its inactivation by PKA leads to aberrant ß-catenin accumulation and associated proliferative signalling.

It should be noted, however, that this inhibition of GSK3ß has been challenged by other groups, which suggest that PGE2-derived accumulation of ß-catenin may be through direct phosphorylation of tyrosine residues, mediated by transactivation of growth factor receptors (139). This mechanism of direct ß-catenin phosphorylation has also been attributed to PKA, as it has been shown to phosphorylate ß-catenin itself at Serine 675, stabilising it (77) Though the functions of these proteins in relation to their interaction with the PGE2 signalling pathway and activation of Wnt signalling has been made clear, it remains to be determined whether the mechanisms for disrupting this pathway are mutually exclusive or if they can all occur in response to PGE2 signalling. More work using specific colorectal cancer cell lines with components of this pathway knocked out must be done to confirm which processes are more prevalent than others.

In addition to the above mechanisms of Wnt signalling activation, PGE2 has been shown to induce activation of this pathway through additional means. When PGE2 signals through EP2, the G protein coupled to that receptor undergoes a conformational change. The α subunit becomes active, exchanging GDP for GTP, allowing it to carry out its function (140). It has recently been shown that the GαS subunit interacts with Axin, an element of the destruction complex (79). The destruction complex is interrupted by this process and ß-catenin is free to accumulate and modulate its

45 proliferative signalling. A role has also been attributed to the γß subunits of the G protein coupled to the receptor. This work showed that these subunits can activate the membrane-bound kinase Phosphatidylinositol-3-Kinase (PI3K) (79). This leads to an inhibitory phosphorylation of GSK3ß and aberrant accumulation of ß-catenin, in a similar manner to that achieved by PKA discussed earlier (79). This group suggested that this phosphorylation was achieved through activation of protein kinase B (PKB, also known as Akt), which plays numerous roles in cellular proliferation (141) and inherently plays multiple roles in tumourigenesis if aberrantly activated (142, 143).

Through the elucidation of ß-catenin:TCF/LEF target genes, which include genes with clear proliferative functions such as c-MYC (144) and CCND1 (Cyclin D1) (145), it has been established that the expression of COX-2 itself can be modulated by ß-catenin signalling (116). This suggests a potential positive feedback loop in which aberrant Wnt signalling and COX-2 overexpression are intrinsically linked and regulate each other. The existence of this signalling loop highlights the potential importance of COX-2 inhibition in the prevention of colorectal tumourigenesis.

With further studies suggesting that COX-2 derived PGE2 may be responsible for the transactivation of growth factor receptors and aberrant activation of their associated growth pathways (80, 139), it may be the case that these mechanisms of modulating cellular proliferation may work synergistically in developing adenomas. With COX-2 ascribed a role in mediating these processes, its inhibition is an important target in cancer chemoprevention.

However, since modulation of proliferative pathways combine with an insensitivity to anti-growth signals in the generation of cancers, determining the roles played by COX-2 in this latter process is important in understanding how adenomas develop and can be prevented.

46 3.3 Tumourigenic Functions of COX-2/PGE2 Signalling – Decreased Sensitivity to Anti-Growth Signals The increasing imbalance of cell growth is crucial during tumourigenesis. While, through mechanisms discussed previously, increased proliferative potential plays an important role, the loss of the ability of a cell to respond to exogenous signals that limit growth is also an important characteristic that shifts the proliferative balance of the cell (130). COX-2 has been shown to play a role in this characteristic in addition to its numerous roles in modulating cellular proliferation, discussed previously.

One of the main anti-proliferative signalling molecules that affects cellular proliferation is Transforming Growth Factor ß (TGF-ß) (146) (see Figure 10). The main pathway of TGF-β induced anti-proliferative effects is the inhibition of components that regulate the cell cycle, forcing cells to withdraw from it, into a quiescent phase known as G0 (146). Hence, TGF-ß signalling confers an antitumourigenic effect, as cells are removed from their proliferative state. Hence, it is advantageous for a cancer if cells that are resistant to TGF-ß signalling are selected for (147). TGF-ß-mediated quiescence is attained both through the inhibition of proteins with roles in cellular proliferation, such as c-myc , and through promoting the production of proteins such as p15INK4b (commonly known as p16) which controls progression through the cell cycle.

This mechanism provides an interesting link to COX-2 overexpression, since COX-2 signalling has been linked to the down-regulation of the TGF-ß Type II receptor (81). Therefore, a dual system may exist to confer tumourigenesis in response to COX-2 overexpression, whereby PGE2 signalling leads to increased levels of c-myc, cyclin D1 and other proliferative proteins through activation of Wnt other proliferative pathways, and the relieving of the inhibition of these proteins by down-regulation of TGF-ß signalling.

47

Figure 10 – The role of TGF-ß signalling in cancer In normal cells, TGF-ß signalling plays a homeostatic role, which involves stalling proliferation of cells through the increased production of genes such as CDKN1A (p21) and the suppression of C-MYC. Upon an early oncogenic mutation, such as the loss of APC, TGF-ß signalling has a tumour suppressive activity. As tumours progress, there is a selection for cells which have lost components of the signalling pathway through further mutations or through down-regulation (147). COX-2 overexpression is associated with down-regulation of the TGF-ß receptor (81). Hence, the cytostatic and pro-apoptotic functions of TGF-ß signalling is lost in these cells.

Image from Massague et al (147), with alterations.

Furthermore, TGF-ß signalling is intimately linked to the onset of colorectal cancer, as the TGF-ß receptor has been shown to be mutated and inactivated in a high proportion of colorectal cancers that arise through the micro-satellite instability pathway (148). Indeed, the importance of an intact TGF-ß receptor, and hence a healthy system of anti-proliferative TGF-ß signalling, in regard to the prevention of tumourigenesis is highlighted by mouse models in which the TGF-ß receptor is knocked out (149).

TGF-ß signalling is intrinsically associated with mechanisms of cellular apoptosis. Various mechanisms of apoptosis are utilised to clear pre-malignant cells. Hence, cancer cells that gain resistance to apoptotic signals have a significant selective advantage. COX-2 has also been linked to this resistance.

48 3.4 Tumourigenic Functions of COX-2/PGE2 Signalling – Suppression of Apoptosis A large body of evidence suggests a role for COX-2 mediated suppression of apoptosis. Insensitivity to pro-apoptotic signals often occurs during tumourigenesis and is considered to be an obligate process in the onset of cancer (150, 151). It is natural to suggest that COX-2 may play roles in the suppression of apoptosis, as without this activity the cell would respond to its modulation of cellular growth with in-built control mechanisms, such as oncogene-induced senescence.

Specifically inducing apoptosis is a crucial mechanism in cancer chemoprevention, as clearing premalignant cells would reduce the risk of developing cancer. A number of early studies suggested COX-2 overexpression may be related to the suppression of apoptosis. The addition of exogenous COX-2 to rat epithelial cells confers increased levels of the anti-apoptotic, pro-survival protein Bcl-2 (81). In addition, these cells also became unresponsive to butyrate induced apoptosis.

Furthermore, the aberrant activation of mitogenic signalling mediated by COX-2/PGE2 signalling, which occurs through mechanisms discussed previously, is clearly associated with the suppression of cellular apoptosis. It has been suggested that aberrant Akt activity, which can occur as a result of PGE2 signalling through its receptors, may be partly responsible for the suppression of apoptosis seen in colorectal adenomas (152-154).

Recent work has highlighted the importance of PGE2 signalling in relation to the suppression of apoptosis. It has recently been suggested that PGE2 may suppress apoptosis through the inhibition of Bim (82) (see Figure 11). This pro-apoptotic ‘BH3 only’ protein contributes to the intrinsic pathway of apoptosis through the activation of other pro-apoptotic proteins, such as Bax and Bak (155), to facilitate permeabilisation of the mitochondrial outer membrane leading to release of cytochrome C and other pro-apoptotic factors (156). In a recent study, it was elucidated that activation of the Ras growth pathway by PGE2, through mechanisms discussed earlier, lead to a marked decrease in apoptosis (82). Both the levels of downstream effectors of apoptosis, such as the cleaved form of

49 caspase 3, and the upstream regulators, such as the predominant isoform of Bim, were decreased in cells treated with high levels of PGE2 (82). The importance of PGE2 signalling in this system of resistance to apoptosis highlights a potential role for drugs that target it. In vitro studies found that the COX-2 selective inhibitors NS-398 and rofecoxib increased levels of Bim and decreased the levels of active ERK1/2 in colorectal cancer cell lines. These findings suggest that the chemopreventative effects mediated by aspirin and other NSAIDs may be in part due to the induction of Bim and hence the restoration of deregulated apoptotic mechanisms. Since the short-term benefits of aspirin have recently been suggested (62), it may also be the case that induction of apoptosis in burgeoning adenomas prevents their growth and provide a short term chemopreventative effect with NSAID prophylaxis.

Figure 11 – Activation of the MAPK cascade leads to degradation of BimEL It has been classically established that PGE2 signalling can transactivate growth factor receptors and leads to aberrant activation of MAPK cascades. It has been shown recently that growth pathways activation leads to phosphorylation of the pro-apoptotic BH3-only protein Bim, which is then targeted to a proteasomal degradation pathway (157). The finding that PGE2 can induce this mechanism of degradation elegantly combines these two processes (82). This may provide an explanation for the induction of apoptosis by NSAIDs and could be the basis for the short-term ‘anti-tumour’ activity of NSAID prophylaxis.

50 This finding concerning Bim was also observed in vivo by both this study and others, with around 40% of colorectal cancers studied displaying decreased Bim expression (82, 158), a finding suggesting that loss of Bim may be a key selective advantage in colorectal cancers. Indeed, this advantage is highlighted by a recent study that identified that Bim plays a key role in TGF-ß mediated induction of apoptosis of colorectal adenomas (159), so loss of Bim – in addition to the mechanisms of TGF-ß receptor loss described earlier – would confer a significant advantage to cancer cells.

Furthermore, mechanisms of COX-2-derived, PGE2-independent mechanisms of resistance to apoptosis have been suggested. A side-effect of COX-2 inhibition is the accumulation of the COX substrate, AA. This accumulation can lead to the production of ceramide (160), which has been shown in to bring about apoptosis in colorectal cancer cell lines (161).

COX-2 has bene intimately linked to additional processes involved in tumourigenesis, inhibition of which may be beneficial at relatively later stages of cancer development.

51 3.5 Tumourigenic Functions of COX-2/PGE2 Signalling – Sustained Angiogenesis While the previously discussed mechanisms of chemoprevention involve the inhibition of processes that lead to the development of adenomas at an extremely early stage, the inhibition of processes that occur once a tumour is beginning to establish itself are also of interest. Ensuring this would theoretically slow the onset and spread of overt cancer, allowing for a better chance of detecting and removing a growing tumour in situ.

Angiogenesis is a critical process in tumourigenesis, particularly in mediating adaptation to potentially deleterious environments, to be discussed later, and enhancing the potential for metastasis. It has been suggested that the inhibition of angiogenesis and metastasis may be the basis for the short term benefit from NSAID prophylaxis (60, 62), which may be preferable given the adverse effects associated with regular use.

Early on in tumourigenesis, tumours will outgrow their local vasculature and will require further blood vessels to grow if they are to grow larger; without the growth of new vasculature tumours would be unable to both receive the nutrients they require and remove the potentially toxic waste they generate (150, 162). The stimulation of angiogenesis is controlled by the relative concentrations of pro and anti-angiogenic factors (130). If the level of pro-angiogenic factors increases above the level of anti-angiogenic factors, angiogenesis will be stimulated.

The mechanism for COX-2 / PGE2 induced angiogenesis has been suggested to be via increased production of Hypoxia Induced Factor 1 (HIF-1)-α and activity of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) (163-165). The concentration of HIF-1α normally acts as the rate-limiting step in the action of the HIF-1 transcription factor, as while HIF-1ß is constitutively expressed in many tissues (166), HIF-1α is rapidly degraded in conditions of normoxia by proteins such as von Hippel-Lindau (VHL) (167) (see Figure 12). In hypoxic conditions, when the growth of new vasculature would be beneficial to a tissue, as it would receive greater oxygen supply, HIF-1α

52 becomes stabilised and binds to HIF-1ß (168). This complete HIF-1 complex acts as a transcription factor at HIF responsive elements (HREs), found in the promoter regions of multiple genes, including VEGF (169).

Figure 12 – The stabilization of HIF-1α during hypoxia The HIF-1 transcription factor binds to HREs in the promoter regions of HIF responsive genes, which include Vascular Endothelial Growth Factor (VEGF) (170) and Basic Fibroblast Growth Factor (bFGF) (171). During normoxia, HIF-1α is hydroxylated by prolyl hydroxylase (PHD), before being ubiquitinated by VHL and degraded. PHD cannot function during hypoxia and hence HIF-1α can bind HIF-1ß. The complete HIF-1 transcription factor binds HREs and modulators of active chromatin.

COX-2 overexpression has been causally linked to an increased expression of pro-angiogenic factors (172), the most common of which in humans are VEGF and Basic Fibroblast Growth Factor (bFGF) . It has also been suggested that both NSAIDs can reduce this production of pro-angiogenic factors (172). This relationship between COX-2 overexpression and VEGF production has been shown in human cell lines, whose expression of VEGF was measured in response to transfection with a COX-2 overexpressing vector (173). Furthermore, addition of PGE2 to cells previously treated with COX-2 selective NSAIDs restored angiogenesis, further suggesting that this signalling pathway was responsible for mediating this process (174). Furthermore, this group suggested that activation of

53 angiogenesis by this mechanism involved activation of mitogenic pathways, culminating in activation of the ERK2 (also referred to as mitogen activated protein kinase MAPK) kinase (174).

A recent study has elucidated another potential mechanism for PGE2-mediated activation of angiogenesis that utilises the MAPK pathway. Though established in a renal environment, this group suggested that activation of this mitogenic pathway, eventually resulting in ERK activation and nuclear translocation, occurred due to PGE2 interacting with intracellular EP receptors (175). This resulted in downstream production HIF-1Îą (175).

While COX-2/PGE2 signalling has been shown to contribute directly to angiogenesis in previous studies, recent work has highlighted a mechanism in which there may be cross-talk between other PGE2 signalling pathways and HIF signalling in the adaption of a tumour to its microenvironment (86, 87), to be discussed later.

3.6 Tumourigenic Functions of COX-2/PGE2 Signalling â&#x20AC;&#x201C; Increased Metastatic Potential Tissue invasion and metastasis is one of the key hallmarks of a cancerous cell (130). The ability to survive without cell-cell or cell-Extra Cellular Matrix (ECM) contacts, digest surrounding structures and intravasate through basal membranes into blood vessels and lymphatics, before spreading to other tissues represents one of the crucial stages of tumourigenesis (150). Prevention of metastasis is crucial, as the main cause of cancer deaths is often not the primary tumour, but rather the spreading of that cancer to distal tissues (176). Hence, characterising mechanisms involved in metastasis that can be inhibited by NSAIDs may be of significant prophylactic and perhaps therapeutic benefit. The importance of preventing metastasis in cancer chemoprevention has been observed in clinical trials. It has been suggested that inhibition of metastasis of established lesions may provide benefits to short-term NSAID prophylaxis (62), which would be preferable given the toxicity associated with regular NSAID use.

54 COX-2 overexpression is causally linked to enhanced motility and invasive potential of cancer cells.

Its importance is highlighted by studies that determined the effect of COX-2 inhibitors on invasive potential of cancer cell lines (83, 84). Both in vitro and in vivo studies have determined that inhibition of COX-2 correlated with decreased metastatic potential. Some groups have highlighted potential mechanisms for these phenotypic changes. It has been suggested that PGE2 derived PI3K / Akt activity, which has also been associated with mediating COX-2 derived proliferative signals, as discussed previously, can increase cellular motility 2.5 fold in a human colorectal cancer cell line (85), visualised by increased invasion through MatrigelÂŽ.

Additional mechanisms have been suggested for this activity. It has been suggested that the COX-2 inhibitor rofecoxib may decrease the viability of metastases once they have been established due to inhibition of angiogenesis (83). This theory is based on a framework of evidence that suggests high serum levels of VEGF, and hence enhanced levels of angiogenesis, are associated with both increased numbers of metastases (177) and a poorer prognosis (178).

It has also been suggested that the COX-2 inhibitor etodolac achieved its suppression of colorectal cancer metastasis through the inhibition of Matrix Metalloprotease 9 (MMP-9) (84), an enzyme involved in the degradation of the ECM (179) â&#x20AC;&#x201C; a process that it is crucial in the metastasis of many cancers. Furthermore, Ă&#x;-catenin, whose deregulation has been discussed previously, has been shown to activate production of other MMPs (180, 181), further emphasising the importance of both these enzymes in metastasis and of PGE2 in regulating their production.

In addition to this, PGE2 signalling has been linked to an invasive phenotype through its transactivation of growth factor receptors (139). Of specific importance is the transactivation of cmet (Hepatocyte Growth Factor). Transactivation of c-met has been shown to be mediated in an EGFR-dependant mechanism (139). This suggests a causal link with PGE2 signalling, since PGE2 has previously been shown to transactivate EGFR (80). This activation of c-met is associated with markedly increased invasiveness of colorectal cancer cells and an increased expression of Urokinase

55 Type Plasminogen Activator (uPAR) Receptor (139). This uPAR system has been intrinsically linked with cancer metastasis, due to its involvement in ECM degradation (182). Additionally, this group determined that COX-2, β-catenin and c-met are all localised at the invasive front of metastatic colorectal cancer cells, implying a degree of interaction between these proteins (139).

As previously mentioned, PGE2 signalling is associated with significant up-regulation of HIF-1. This transcription factor has been termed a “master regulator” due to its numerous, multi-faceted target genes (183). These have been shown to include proteins involved in invasion of cancer cells (183), including the uPAR system described previously, MMP-2, which has been shown to be involved in colorectal carcinogenesis (184, 185) and cathepsin D, a protease also characterised in colorectal cancer invasion (186). As such, HIF-1α – and hence HIF-1 – over-activity has been shown to be associated with a decreased likelihood of cancer patient survival (187).

COX-2/PGE2 signalling has been highly implicated in tumour invasiveness and metastasis, and is particularly involved in cross-talk between the other hallmark characteristics of cancer, such as sustained angiogenesis. The role of COX-2/PGE2 signalling in the invasiveness of cancer cells may determine the potential use of COX-2 inhibitors as adjuvant therapies, as reducing the generation and viability of metastases could significantly improve cancer survival.

Work is continuing to determine whether NSAIDs have additional activities at slightly later stages of tumourigenesis, such as preventing the adaptation of cells to the rapidly changing tumour microenvironment. While these activities may not provide primary chemoprevention, they may still be clinically relevant as preventing the enhanced growth of established adenomas and burgeoning tumours may prevent development of overt disease and the spread of the cancer to distal tissues.

56 3.7 Tumourigenic Functions of COX-2/PGE2 Signalling – Adaptation to the Tumour Microenvironment The adaptation of cancer cells to increasingly cytotoxic environments is a crucial target in the prevention of cancer development, as it may prevent the growth of tumours and could provide therapeutic benefit. Potentially deleterious conditions, such as hypoxia, an environment encountered by all tumours that outgrow their local vasculature, provide a selective pressure that initiates a process of clonal evolution. If cells do not adapt to this environment hypoxia can lead to the induction of cell death (188). Adaptation ensures that cancer cells can survive in these environments. Hence, prophylactic and therapeutic intervention at this stage is an attractive and potentially crucial proposition, as it would in theory prevent tumours from growing beyond a very small size. Further, COX-2 can be both regulated by and regulate the interaction of cancer cells to their environment. Hence, inhibition of this isoform in particular is of great interest.

Since adaptation to a hypoxic environment is a crucial process in tumour development and COX-2 overexpression is both intrinsically linked to colorectal tumourigenesis and found at increasing levels as tumours grow (91), a relatively recent study set out to determine whether these processes were related and whether COX-2 overexpression may be a downstream result of HIF-1 activation, an obligate process during hypoxia (86). This study found that hypoxia resulted in an increase in COX-2 expression in colorectal cancer cell lines. It was reported that HIF-1 binding to the COX-2 promoter was responsible for this increase, which also resulted in increased levels of COX-2 derived PGE2. This PGE2 was shown to be responsible for both survival of these cells during hypoxia and an enhanced level of HIF-1 activity (see Figure 13). It was suggested that the latter activity mediated through modulation of the MEK/ERK pathway – a function already attributed to PGE2 signalling by others during its function in normoxia (80).

This same group suggested a mechanism for this COX-2 derived survival during hypoxia. This group suggested that ß-catenin may switch its target transcription factor during hypoxia, from TCF to HIF-1 (87). This has two main functions: it relieves the proliferative functions of the ß-catenin/TCF

57 complex, ensuring that the cell is not proliferating during potentially deleterious environments; it also increases the rate of HIF-1 mediated adaptation to a hypoxic environment, through for example the stimulation of VEGF production (169). This may have wide-ranging clinical implications. Since many cancer therapies rely on the fact that these cells are actively dividing and the adaptation of cancer cells to hypoxic environments results in a decrease in cellular proliferation, this process may render such cells resistant to chemotherapy. Inhibition of this adaptation through inhibition of COX2 may be of substantial therapeutic benefit, sensitising cancer cells to both cytotoxic environments and chemotherapy.

Therefore, there may be reason to suggest that based on these findings that inhibition of COX-2 may play a role in the suppression of adaptation to potentially deleterious environments. This exploitation of the cancer microenvironment is a burgeoning field, with a number of treatments already being considered that capitalize on the hypoxic environment cancer cells find themselves in (189). However, treatment using this groupâ&#x20AC;&#x2122;s findings would represent a new development, through direct inhibition of the cancer cellâ&#x20AC;&#x2122;s adaptation to that environment.

58

Figure 13 – There is cross-talk between numerous pathways in the adaptation of a cancer cell to hypoxia It has been reported that the HIF-1 transcription factor, whose activity is activated during hypoxia, results in increased levels of COX-2 derived PGE2. COX-2, through classically established mechanisms, leads to enhanced β-catenin activity. However, in a positive feedback loop, β-catenin is thought to enhance expression of HIF-1α, rather than activating TCF/LEF, during hypoxia.

In addition to hypoxic stress, cancer cells can also be subjected to additional stresses to which they also need to adapt in order to survive. Recent work has highlighted an interesting relationship between Wnt signalling, one of the key signalling pathways discussed throughout this dissertation, and resistance to autophagy. Autophagy is a key homeostatic process that mediates the quality control of the intracellular environment and facilitates the adaptation of cells to environments in which nutrients have become scarce and the cell must recycle its own contents for energy (190) (See Figure 14). As such, it can be seen to act as both a tumour suppressive process, by reducing the

59 viability of potentially mutagenic substances such as reactive oxygen species and toxic protein fragments, and a tumour promoting process, since evidence exists that it can be aberrantly activated in the adaption of tumour cells to potentially deleterious environments such as nutritional stress (191).

Figure 14 â&#x20AC;&#x201C; Autophagy Autophagy is an important process in the homeostatic degradation of damaged proteins and organelles and proteins targeted for degradation by ubiquitination and proteins prone to aggregation (192, 193). Two complexes mediate the induction of the process, including the ULK1 complex and the PI3KC3 complex. These attract autophagy (Atg) proteins, which form complexes that lead to the conjugation of lipids to a membrane isolated from existing cellular components, forming the autophagosome. The completed autophagosome fuses with the lysosome to form the autophagolysosome and through the action of hydrolases and permeases, the engulfed contents are degraded and recycled (194).

The importance of autophagy in colorectal tumourigenesis has been highlighted by the discovery of an interaction between Wnt signalling and the autophagic process (88). Ă&#x;-catenin, the key modulator of the Wnt signalling pathway, which becomes constitutively active in a large proportion of colorectal cancers, was shown in this study to be a negative regular of the pro-autophagic protein

60 p62, which is involved in targeting substances to be degraded to the lysosome (195). However, in a seemingly conflicting finding, ß-catenin was also found to bind components of the autophagic machinery, suggesting that ß-catenin may be degraded by the autophagosome; a pathway independent of the traditional destruction-complex mediated proteasomal mechanism.

These findings suggested that autophagy is both induced and repressed in cancer, depending on the context of the tumour environment. Through suppression of p62, ß-catenin suppresses basal levels of autophagy, enhancing tumourigenicity. However, upon the induction of nutritional stress once the tumour has outgrown its nutrient supply, ß-catenin function is switched away from activation of TCF signalling, as proliferation during nutritional stress would be deleterious to the cell. This mediates ß-catenin degradation by the autophagosome (88). Additionally, this relieves the inhibition of autophagy, allowing the cell to adapt to nutritional stress.

The potential therapeutic exploitation of the complex roles of autophagy in tumourigenesis has been investigated in recent years. It has been suggested that aspirin both inhibits Mammalian Target of Rapamycin (mTOR) signalling and activates AMP-activated kinase (AMPK) and that both of these mechanisms result in enhanced autophagy and, interestingly, induction of apoptosis in colorectal cancer cells (196) (See Figure 15). These pathways that regulate autophagy have been intrinsically linked to colorectal tumourigenesis through additional targets. Targets of mTORC1, such as P70-S6 Kinase 1 and Eukaryotic translation initiation factor 4E-binding protein 1 (EIF4e-BP1) (197), have been shown to be overexpressed in colorectal cancer (198). Furthermore, inhibition of mTOR has been shown to decrease the number of polyps formed in Min mice (199), implying that mTOR is an attractive, multi-faceted target for chemoprevention.

The complex roles of autophagy in cancer mean that significant work is required to elucidate exactly how the process can be targeted therapeutically. This is highlighted by the varying approaches regarding cancer therapy involving autophagy: both the induction of autophagy, through the

61 inhibition of proteins such as mTOR, and its inhibition, suggested to reduce chemoresistance, have been suggested as beneficial strategies (200).

Figure 15 â&#x20AC;&#x201C; Aspirin modulates the regulation of autophagy Through the inhibition of mTOR and activation of AMPK, aspirin leads to the activation of the ULK complex and hence activation of autophagy in burgeoning cancer cells (196). Activating autophagy can either reduce the viability of tumourigenic substances, or if induced at a high enough level, may induce apoptosis in cancer cells.

Inhibition of mTOR also has additional anti-tumourigenic functions (197)

The adaption of cancer cells to the tumour microenvironment is now beginning to be seen as a crucial process in tumourigenesis. It allows for survival in potentially deleterious environments, which act as significant selection pressures, and increases the viability of distal metastases. Various mechanisms have been suggested for this adaption and NSAIDs, through both COX-dependent and independent mechanisms, have been suggested as a therapeutic strategy to prevent this adaption.

62 The use of NSAIDs as therapeutic strategies is gaining significant interest. In addition to its roles in driving the early stages of tumourigenesis, the COX-2/PGE2 signalling pathway has been implicated in processes that are thought to occur throughout cancer development, including the maintenance of putative cancer stem cells. Hence, NSAIDs may be clinically relevant throughout the timeline of cancer development.

3.8 Tumourigenic Functions of COX-2/PGE2 Signalling â&#x20AC;&#x201C; Maintenance of Stemness Stem cells are thought to play a role in the maintenance of certain adult tissues. Through their markedly increased proliferative potential, ability to self-renew and ability to generate heterogeneous progeny cells, stem cells are placed at the apex of the developmental hierarchies of many tissues (201). Similarly, a large body of evidence suggests that similar cells may be responsible for feeding tumours with new cancer cells. These â&#x20AC;&#x2DC;cancer stem cellsâ&#x20AC;&#x2122; share the three main characteristics of adult stem cells and are thought to be responsible for producing the significant heterogeneity seen amongst cells in a tumour (202). As well as feeding the tumour, cancer stem cells are also thought to function as chemorefractory centres that allow for cancers to relapse after seemingly successful treatment and as the cell of origin for distal metastases (202).

It is clear, then, that clearance of cancer stem cells is a key target for preventative and therapeutic strategies. The need for targeted clearance is significant, as total remission may not occur without the removal of such cells that continually maintain the tumour. The elucidation of cell surface markers intrinsically associated with both normal and cancer stem cells longed remained elusive.

However, a crucial study potentially identified Leucine-rich repeat containing G-protein coupled receptor 5 (LGR5) as such a marker (203) and subsequent studies have corroborated these findings (204) and putatively identified LGR5+ stem cells in other tissues (205). LGR5 has been shown to have a functional role in maintaining the stemness of cells in the colonic crypt, since LGR5+ cells can reconstitute structures resembling crypts in culture (206) and it has been shown that loss of the APC in these putative stem cells, rather than any other cells present in the colonic crypt, is sufficient to

63 generate adenomas in mice models (207). Indeed, in line with marking cancer stem cells, recent studies have linked enhanced LGR5 expression with a poor response to chemotherapy (208).

Since Wnt signalling, as discussed previously, has been shown to be highly active at the base of intestinal crypts (131), it was hypothesised that LGR5 was a Wnt target gene. A further level of complexity is highlighted by the view that LGR5 acts to potentiate Wnt signalling and inhibit antagonists of this signalling, highlighting a potential positive feedback loop in the maintenance of the stemness of cells at the base of the crypt (209, 210). Two principal mechanisms have been attributed to LGR5 in the maintenance of stemness. One study generated a model of an interaction between LGR5 and the Wnt signalling components Low-density lipoprotein receptor-related protein 6 (LRP6) and Frizzled-5 (FZD5), resulting in internalization with these components in a mechanism that potentiates Wnt ligand binding (209). Another linked the potentiation of Wnt signalling to binding of the LGR5 ligand R-spondin to its receptor, resulting in suppression of membrane-bound E3 ubiquitin ligases that act to mediate the destruction of Wnt receptors (210).

Using the discovery that LGR5+ cells were likely to be the cell of origin in colorectal adenomas (211) and that LGR5 itself is a Wnt target gene; a potential role for COX-2/PGE2 signalling in the maintenance of stemness has been hypothesised. A recent study highlighted a potentially key relationship between PGE2 signalling and LGR5 and further elucidated the role of LGR5 in both healthy and cancer stem cells (89). This group suggested that LGR5 expression was highest in adenomas, decreasing as cancers progress through the steps of carcinogenesis (89). In addition, PGE2 was shown to positively regulate LGR5, highlighted by the fact that cells rendered unresponsive to PGE2 did not show increased levels of LGR5 upon addition of exogenous PGE2. LGR5 was also attributed to be a crucial effector of PGE2 mediated cell survival, since enhanced survival resulting from addition of exogenous PGE2 was removed upon the silencing of LGR5 (89).

Findings from additional studies that detail the steps required for the generation of cancer stem cells highlight further potential targets for chemotherapeutic intervention. It has been found that

64 mesenchymal stem cells, upon activation by nearby colorectal carcinoma cells, generate a niche that accommodates carcinoma stem cells, in a mechanism associated with PGE2 release by autocrine activation of these mesenchymal cells (212). This relationship between cancer stem cells and their microenvironment could provide the basis for the use of NSAIDs as adjuvant chemotherapeutic agents. Removal of carcinoma stem cells through the inhibition of this mechanism could prevent the generation of chemorefractory cells and hence decrease the risk of cancer relapse (213).

Indeed, in keeping with the maintenance of putative cancer stem cells, COX-2 has been implicated in mediating cellular characteristics attributable to these cells.

3.9 Tumourigenic Functions of COX-2/PGE2 Signalling â&#x20AC;&#x201C; Resistance to Chemotherapy Resistance to chemotherapy is a significant challenge in the treatment of cancer. Failure to respond to chemotherapy is indicative of a poor prognosis. Hence, preventing the onset of resistance and resensitising cells to therapy would be of substantial therapeutic benefit. A growing body of evidence suggests that COX-2 overexpression may be associated with resistance of cancers to traditional chemotherapy and that hence COX-2 inhibitors may play a role as adjuvant chemotherapeutic agents.

It has been suggested that COX-2 mediates the expression of the gene Multi Drug Resistance Protein 1 (MDR1), which codes for P-glycoprotein (PGP), in colorectal cancer (214). MDR1 has been causally linked to resistance to chemotherapy in a number of cancers (see Figure 16), including colorectal cancer â&#x20AC;&#x201C; though it should be mentioned that a slightly more recent study has suggested that MDR2 may in fact be a key mediator of chemoresistance in colorectal cancer (215).

65

Figure 16 â&#x20AC;&#x201C; PGP mediates resistance to chemotherapy PGP mediates drug efflux through the active transport of molecules through the plasma membrane. This process requires the binding of ATP to specific domains, a mechanism that characterises the ATP-binding cassette (ABC) family of proteins to which PGP belongs. PGP has been shown to mediate resistance to a number of classically used chemotherapies, including Etoposide and Doxorubicin.

Image from Sorrentino et al (216)

Though a combination of COX-2 inhibitors with traditional chemotherapy has previously been shown to be beneficial in the treatment of cancer and the prevention of metastasis (217, 218), a recent study has characterised the relationship between the two processes in some detail. This study determined that combination therapy with aspirin or celecoxib and 5-fluorouracil (5-FU), a common chemotherapeutic agent used in the treatment of colorectal cancer, reduced the viability of colorectal cancer cell lines that were resistant to 5-FU. Crucially, it was determined that the combination therapy achieved this through re-sensitisation to traditional chemotherapy (219), as combination therapy lead to a decrease in both COX-2 and MDR1 levels (219). These effects were observed both in vitro and in vivo, with chemorefractory xenografts showing encouraging regression upon combination therapy, compared to traditional chemotherapy alone (219).

Furthermore, since COX-2 overexpression has potentially been associated with the generation of cancer stem cells, a defining characteristic of which is resistance to chemotherapy (213), it may be the case that there are more and more pathways associated with COX-2/PGE2 signalling that mediate chemoresistance that remain to be elucidated. Though the body of evidence that suggests

66 the use of COX-2 inhibitors as adjuvant therapies is growing, far more work is required to prove the efficacy of this treatment.

At the time of writing there are a number of studies, some of which have been discussed here, that suggest a relationship between COX-2 overexpression and MDR-mediated chemoresistance.

However, very few clinical trials exist that determine how effective combination therapy may be in the treatment of human cancers. One of the few that set out to determine this contained a very small sample size and was ended early due to a large number of non-cancer related deaths (220), potentially due to the inherent risk of adverse cardiovascular events associated with regular COX-2 inhibitor use. As such, although the in vitro and in vivo studies yield promising results, it remains to be proved how effective this combination therapy is in the treatment of human cancers.

3.10 The Role of COX-2 in Tumourigenesis â&#x20AC;&#x201C; Concluding Statements Recent decades have greatly enhanced the understanding of how COX-2 and PGE2 contribute to colorectal tumourigenesis. It is becoming increasingly clear that COX-2 inhibition may derive benefit both in the prevention of polyp development and at later stages in tumourigenesis, preventing tumour growth and potentially clearing populations of tumour initiating cells. Hence COX-2 inhibition may have both prophylactic and therapeutic benefit.

However, with it becoming clear that COX-2 overexpression is not apparent at the earliest stages of tumourigenesis (91) and hence may not fully explain the chemopreventative activities of some NSAIDs, increasing numbers of studies are beginning to explore the putative roles played by COX-1 in colorectal tumourigenesis.

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4. COX-1 in Colorectal Tumourigenesis While a great deal of work regarding the chemopreventative effects of aspirin and other NSAIDs has focused on COX-2 inhibition, there is a growing body of evidence to suggest that COX-1 may aplay a role in tumourigenesis, and hence its inhibition has been explored.

4.1 Early Evidence for COX-1 Involvement in Tumourigenesis A key study in the field of chemopreventative COX-1 inhibition set out to determine whether a particular COX isoform could be causally linked to colorectal tumourigenesis (90). This study, and many others that model colorectal carcinogenesis, used Min mice, which contain an inactivating mutation in one allele of the APC gene (221) and can hence be seen as an animal model for FAP. Consequently, they develop a large number of polyps at an early stage. Regression or prevention of these polyps can be used to determine successful chemoprevention. These mice were crossed with either COX-1 or COX-2 knockout mice (90), producing progeny that had lost both the APC and either of the COX genes. This study reported that knockout of not only COX-2 but also COX-1 in Min mice improved survival and decreased polyp number (90). Furthermore the decrease in polyp number was similar for both knockouts, highlighting that COX-1 may play more of a role in colorectal tumourigenesis than first thought. Furthermore, in addition to these findings, it was later found that treatment of both healthy rats and Min mice with a COX-1 specific inhibitor decreased numbers of both aberrant crypt foci and resulting colorectal adenomas (222). Taken together, these findings suggested that COX-1 inhibition could potentially pose just as promising a chemopreventative target as COX-2 inhibition.

Crucially, though, these studies did not provide data for the effect of COX knockout on polyp size.

The absolute reduction in polyp number, though encouraging and useful data, gives no information as to what stage of tumourigenesis COX-1 plays a role and hence whether it is a feasible target for

68 chemoprevention. Ascertaining exactly what role COX-1 may play in colorectal tumourigenesis is crucial and work continues to this day.

Studies that established the pharmacokinetics of aspirin may retrospectively provide evidence for COX-1 inhibition in explaining the findings of the epidemiological and clinical studies that suggested a chemopreventative effect. It has been determined that aspirin, the basis of numerous studies described previously and shown to have chemopreventative effects, preferentially inhibits the COX-1 isoform (93). While aspirin and some other commonly prescribed NSAIDs can inhibit both COX isoforms at sufficient concentrations, the fact that low-dose aspirin, which is not thought to inhibit COX-2, has been shown to prevent tumourigenesis, it is natural to suggest that inhibition of COX-1 is largely responsible.

Further pharmacokinetic analysis of aspirin has provided further potential evidence for a role of COX-1 in tumourigenesis. It has been reported that the peak concentration of salicylic acid in the plasma, around 2ÎźM (223), is far lower than the IC50 â&#x20AC;&#x201C; the amount of a specific inhibitor that is required to inhibit the activity of its substrate by half â&#x20AC;&#x201C; of aspirin for COX-2, which has been reported at varying levels, though unanimously at levels higher than the concentration of salicylic acid in the plasma (92, 93). It must be emphasised that these studies were not undertaken in the context of cancer research; rather they were undertaken to understand the general pharmacokinetics of aspirin and its effect regarding cardiovascular disease. Hence, these findings can only putatively be applied retrospectively to explain the findings of clinical trials. Further, it should be noted that exploration of pharmacokinetics must involve the potential distinction between whether these agents act on epithelial cells at the site of the tumour or whether COX-1 is largely inhibited pharamacologically at other sites.

69 4.2 Elucidating the Role of COX-1 in Tumourigenesis â&#x20AC;&#x201C; Evidence for Early Involvement Determining the exact role of COX-1 in colorectal tumourigenesis, particularly at what stage it is involved, is extremely important as primary chemoprevention relies on the inhibition of processes that occur at an early stage. Numerous lines of evidence exist to suggest that COX-1 may indeed be involved at an early stage.

A putative early role has been suggested by studies that originally set out to determine COX-2 expression in the various stages of colorectal carcinogenesis. One, upon immunohistochemical staining for COX-2 in colorectal adenomas of differing sizes (91), found that while COX-2 overexpression clearly occurred in the early stages of tumourigenesis, it could not be identified as a causative factor during the earliest stages of adenoma development. It found that only 38% of the small adenomas in the study expressed COX-2, with particularly strong staining occurring in just 8% of these adenomas (91). Though larger adenomas clearly displayed COX-2 overexpression, the lack of consistent overexpression in the smaller adenomas suggested that COX-2 may not be the factor that mediates the earliest stages of tumourigenesis.

Studies that undertook immunohistochemical staining of both normal and colorectal cancer samples suggest that any potential effect of COX-1 in tumourigenesis does not occur due to overexpression (224, 225) and hence may contribute to colorectal tumourigenesis in a different mechanism to COX2. The former of these studies has suggested that COX-1 is in fact down-regulated in colorectal cancer. Interestingly, COX-1 was increasingly down-regulated as carcinogenesis proceeded.

Adenomas saw lower COX-1 levels than normal tissues, but more than carcinomas (224). This implies that if COX-1 has any role in colorectal tumourigenesis, it is likely to be in the earliest stages, before any potential down-regulation. Few studies have corroborated this finding, so the reports of this group must be viewed with a degree of caution. Taken together, these studies suggest that COX-1 may act during a very early stage of tumourigenesis and numerous studies have been undertaken to more precisely characterise the mechanisms by which COX-1 may mediate tumourigenesis.

70 4.3 Elucidating the Role of COX-1 in Tumourigenesis â&#x20AC;&#x201C; Activation of Carcinogens In keeping with a role in the early stages of tumourigenesis, inhibition of COX-1 has been linked by various studies to the protection of the genome from mutagenesis. A relatively early study set out to determine whether the COX enzymes could activate a wide range of known environmental and dietary pro-carcinogens (95). The activation of carcinogens is a crucial process in tumourigenesis, as they tend to exist in inactive forms which must be converted into genotoxic substances before exerting their effects. It has been shown that recombinant human COX-1 (hCOX-1) is able to activate a number of common carcinogens (95), including those which have been causally linked to colorectal tumourigenesis, such as the heterocylic aromatic amines (HAA) (95), a group of carcinogens that are commonly generated by overcooking meat and may therefore play a role in colorectal tumourigenesis once ingested (226). Once activated, HAAs can form DNA adducts, structures that if not repaired correctly can lead to mutation of the DNA (Reviewed in 227).

A recent epidemiological study proposed that intake of PhIP, a common HAA found in red meat and shown to be activated by COX-1 by Wiese et al (95), was associated with colorectal cancer onset (228). Participants allocated to a high dose of red meat saw a 46% increased risk of developing colorectal adenomas (228).

Crucially, COX-1 was also shown to activate the common carcinogen benzo(a)pyrene (B(a)P)-7,8-diol, a metabolite of B(a)P (see Figure 17). B(a)P is a member of the polycyclic aromatic hydrocarbon family of carcinogens, which are particularly pertinent in the tumourigenesis of colorectal cancer (95). B(a)P intake, which can occur from a number of sources such as cigarette smoke (229) and cooked processed meat (230), has been shown to be associated with an increased risk of colorectal cancer (231). The importance of B(a)P metabolism in the carcinogenesis of colorectal cancer is highlighted by the fact that an isoform of cytochrome P450 required for its metabolism into precursor molecules of B(a)P-7,8-diol is overexpressed in colorectal adenomas (232).

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Figure 17 â&#x20AC;&#x201C; B(a)P metabolism B(a)P requires multiple stages of activation before forming a carcinogenic product. Certain cytochrome P450 family members (CYP1A1) and hydrolase enzymes convert B(a)P to B(a)P-7,8-diol. COX-1 has been shown to mediate the conversion of this compound to its final carcinogenic product (95).

Image from Wikipedia commons (233), with alterations.

The inhibition of this carcinogen activating function of COX-1 may play a crucial role in protecting the genome from potentially carcinogenic mutations. Indeed, the work previously discussed may partly explain earlier findings that co-treatment of rats with 1,2 dimethylhydrazine (DMH), a common carcinogen associated with colorectal carcinogenesis, and aspirin reduced the induction of colorectal cancer (234). Additionally, COX-1 has been directly linked to the production of malondialdehyde (MDA), a carcinogen implicated in colorectal tumourigenesis (235), produced through the breakdown of the precursor prostanoids produced by the COX enzymes (236).

Since the addition of environmental carcinogens has been shown to be sufficient to induce carcinogenesis in the small intestines of Min mice that have lost their pre-disposition to polyp generation (237), COX-1 may play a crucial role in tumour initiation and hence may be a feasible target for cancer chemoprevention.

72 It is possible that the loss of COX-1-derived carcinogens such as MDA, or those discussed above; BP7,8-diol and PhIP, may be responsible for the decreased incidence of colorectal polyps in COX-1 knockout mice (90). The Min mouse used by this group and many others is heterozygous for an APC mutation. The seminal â&#x20AC;&#x2DC;two-hit hypothesisâ&#x20AC;&#x2122; states that a second allele of a tumour suppressor gene must be lost for a cancer to progress (238). Therefore, it could be suggested that loss of COX-1 could have prevented the activity of potential carcinogens, preventing the loss of heterozygosity required for carcinogenesis.

However, it must be mentioned that while epidemiological studies suggest that environmental carcinogens are linked to the onset of colorectal cancer (228, 231) and addition of environmental carcinogens has been suggested to be enough to lead to tumour induction (237), it is not yet clear whether the pro-carcinogen activating function of COX-1 is pathologically relevant in vivo. In all likelihood, the mechanisms of somatic aberrations pertaining to colorectal tumourigenesis are not mutually exclusive. Therefore, activation of dietary pro-carcinogens may be an important function in tumourigenesis.

With these functions having been elucidated, it became important to identify a role for COX-1 in directly mediating colorectal tumourigenesis in pathologically relevant in vivo scenarios.

73 4.4 Elucidating the Role of COX-1 in Tumourigenesis - Lessons from Protection of Intestinal Stem Cells Determining the role played by COX-1 in pathological scenarios in the colon will determine the feasibility of COX-1 inhibition in cancer chemoprevention. In keeping with being a good target for chemoprevention, there is a body of evidence that suggests that COX-1 may be involved in the protection of intestinal stem cells. Regulation of the survival of intestinal stem cells, specifically after damage, is crucial as stem cells that undergo carcinogenic mutations could become cancer-initiating cells that have the potential to continuously feed a tumour with heterogeneous cancer cells and became a therapy-refractory population that prevents total cancer clearance (201).

Therefore, while protection of stem cells in normal environments is crucial, as they maintain the rapid turnover that occurs in both intestinal and colonic crypts (239), stem cell survival upon genotoxic damage is no longer a favourable process and characteristic of tumourigenesis. Early work highlighted a potential role for COX-1 in mediating the pro-survival response that protected putative intestinal cancer stem cells upon damage induced by radiation (94). The COX-1 inhibitor indomethacin reduced the number of intestinal stem cells that survived an insult by gamma radiation (94). Furthermore, treatment of COX-1 knockout mice with a known carcinogen, azoxymethane (AOM), a metabolite of DMH, was reported to result in a lack of the pro-survival response that is traditionally seen upon mutation induced by AOM (240).

The findings of these studies may have provided the first suggestion that common mechanisms of tumourigenesis may be partly mediated by COX-1, highlighting that carcinogen and radiation induced tumourigenesis is lost upon its inhibition or inactivation. Interestingly, Cohn et al provided one of the first suggestions that the contribution of COX-1 to tumourigenesis may be mediated through production of PGE2, highlighting an interesting question when comparing COX-2 and COX-1 mediated tumourigenesis: how can both enzymes contribute to tumourigenesis through a similar mechanism when only one is overexpressed?

74 It was becoming clear that COX-1 had activities that mediate colorectal tumourigenesis. Though the exact mechanisms by which it functions remain largely unknown, numerous studies are now being undertaken in order to shed light on its role as understanding the exact mechanisms by which COX-1 contributes to colorectal tumourigenesis informs the feasibility of COX-1 as a target in chemoprevention and allows targeted manipulation of the pathways in which it is involved.

4.5 Elucidating the Role of COX-1 in Tumourigenesis - First Suggestions of a Mechanism for COX-1 Mediated Tumourigenesis Given these considerations, recent work has identified a novel interaction between the Wnt signalling pathway and the control of PGE2 turnover as being potentially important in COX-1 mediated tumourigenesis (241). The Wnt signalling pathway has been discussed previously in this dissertation. Aberrant activation of this pathway through the effects of PGE2 (77, 79, 138, 242) has been associated with colorectal tumourigenesis. Hence prostaglandin synthesis by COX enzymes has gained a great deal of interest.

However, until recently very little work had been done regarding the turnover of PGE2. 15Prostaglandin Dehydrogenase (15-PGDH) has previously been identified as playing a role in PGE2 turnover (243). It catalyses its conversion to 15-keto-PGE2 (see Figure 18), which has a decreased activity compared to its native counterpart and is the first step in its degradation. 15-PGDH has previously been putatively identified as a tumour suppressor gene, as it is down-regulated in many cancers (244-247) and 15-PGDH knockout has been shown to increase the onset of tumours in Min mice and enhance the generation of adenomas initiated by AOM treatment (248). However, though these earlier studies identified 15-PGDH as a tumour suppressor and suggested its regulation of PGE2 levels as a possible explanation for this, these studies largely came to the same conclusion as the majority of the literature at the time; that only COX-2 was the source of the PGE2 that is inactivated by 15-PGDH. However, since most of the groups investigating 15-PGDH were performing studies concerning the very initial stages of colorectal tumourigenesis, even at the aberrant crypt foci level, it was unlikely that COX-2 was responsible for these levels. Since COX-2 overexpression had been

75 previously identified as a somewhat later event in tumourigenesis (91), the cause of the enhanced PGE2 levels upon 15-PGDH loss remained unidentified.

Figure 18 – 15-PGDH couples with PGE2 influx with inactivation and degradation While PGE2 influx through EP receptors results in numerous signalling processes that have been linked to tumourigenesis, influx through other cell surface molecules, such as PGT (126), couples PGE2 influx with its degradation. After this first stage of PGDH inactivation, 15-PGDH is thought to be responsible for the conversion of PGE2 into an inactive form, 15-keto-PGE2. This form also acts as the substrate for PGE 2 degradation and turnover. Hence, 15-PGDH can be seen as antagonistic to COX-2 signalling, as the former is responsible for PGE2 catabolism while the latter mediates its metabolism. PGEM – PGE2 metabolite.

The study by Smartt et al highlighted a potentially novel role for COX-1 derived PGE2 in colorectal tumourigenesis, providing a potential answer to the questions raised by previous early studies (see ‘5.4 Elucidating a role for COX-1 in tumourigenesis: lessons from protection of intestinal stem cells’).

Through its discovery that ß-catenin negatively regulates the expression of 15-PGDH in a TCFdependent mechanism (241), this group elegantly identified a putative positive feedback loop that incorporates both COX-1 and COX-2 derived PGE2 and Wnt signalling (see Figure 19). This group suggested that since APC mutation and hence aberrant ß-catenin activation is often the initiating

76 event in colorectal tumourigenesis, suppression of 15-PGDH activity and the accumulation of basal PGE2 produced by the constitutively expressed COX-1 that follows may be important in mediating colorectal tumourigenesis. PGE2 has been shown to further stabilise and activate ß-catenin in previous studies and hence a positive feedback loop is generated. Since ß-catenin is also thought to positively regulate COX-2 expression, it may be the case that the mechanism suggested by Smartt et al may be extended to incorporate the COX-2 overexpression seen in later stages of adenoma development.

This was among the first studies to incorporate 15-PGDH into a potential mechanism for COX-1mediated colorectal tumourigenesis and may provide a basis for further investigation.

Figure 19 – ß-catenin negatively regulates 15-PGDH expression Mutation of the APC gene is one of the earliest events in colorectal tumourigenesis. This leads to aberrant ß-catenin activity. With it being shown that ß-catenin negatively regulates 15-PGDH expression (241), it has been suggested that this may mediate a positive feedback loop in which COX-1 derived PGE2 has both tumourigenic effects and leads to further activation of ß-catenin activity.

77 The identification of a second mechanism for the regulation of PGE2 levels raised a number of questions regarding the putative model of COX-2 derived, PGE-2 mediated colorectal tumourigenesis. Since 15-PGDH is lost in very small adenomas, it has even been suggested that it may be lost in single aberrant crypt foci (248), its loss is thought to a play a key role in tumour initiation or very early tumourigenesis. Furthermore, COX-2 overexpression is rarely detectable in adenomas smaller than 5mm (91). Hence, it is unlikely that the PGE2 derived from overexpressed COX-2 is the source of the increased PGE2 levels detected upon loss of 15-PGDH. Taken together, the findings that COX-1 may enhance the mutagenic activity of environmental procarcinogens and that 15-PGDH is lost at an early stage of tumourigenesis, before COX-2 overexpression tends to occur, suggest that COX-1, may play a key role in the early stages of colorectal tumourigenesis. Through these activities, COX-1 may be responsible for both the transformation of normal cells and progression of these transformed cells into a more malignant state.

Following on from these recent findings, 15-PGDH is beginning to receive significant investigation. A recent study investigated the expression of 15-PGDH in colonic samples and determined that not only do 15-PGDH levels remain constant throughout the colon, they also remain at a similar level over time (249). These stable expression patterns highlight the importance of the protein in the maintenance of prostaglandin levels in the normal colon. This study also highlighted a significant level of variation of 15-PGDH levels between individuals (249). It is not yet known whether the lower end of the expression spectrum is low enough to be a risk factor for the induction of colorectal tumourigenesis through COX-1 derived PGE2 accumulation and signalling. Intrinsically low 15-PGDH levels, however, have been suggested as a mechanism of resistance to treatment with COX-2 selective inhibitors such as celecoxib (250). Further, single nucleotide polymorphisms in the 15PGDH gene have also been suggested to be responsible for poor response to COX-2 inhibitors and as risk factors for developing colorectal cancer (251). Since 15-PGDH may be seen as having the

78 potential to reduce the effectiveness of COX-2 inhibitors, it may be the case that these individuals may benefit more from the chemopreventative effects of COX-1 inhibition.

4.6 Lessons from Investigations into COX-1 - the Significance of 15-PGDH in Colorectal Cancer With a potential mechanism now ascribed to the contribution of COX-1 to colorectal tumourigenesis, components of this pathway are worth investigating to characterise colorectal cancer risk factors and to stratify individuals to specific chemopreventative strategies.

Recently, stimulating 15-PGDH expression has gained significant attention (252) and these studies may provide the basis for further chemopreventative exploration in the future. The overexpression of pro-inflammatory cytokines is thought to mediate chronic inflammatory disorders such as IBD, a classically defined risk factor for colorectal cancer (27). A putative explanation for the oncogenic effect of these cytokines, as well as the induction of COX-2 activity (51), is the suppression of 15PGDH activity (253), a process that can also occur through the activity of other compounds produced during inflammatory conditions (254). 15-PGDH downregulation has also been associated with other molecular pathways involved in colorectal cancer. It has been shown to be a positively regulated product of TGF-ß signalling (255), which can be lost in a number of ways in colorectal cancer; either through mutations in effector molecules of the pathway (256) or through loss of the TGF-ß receptor itself through aberrant CpG island methylation or as a result of microsatellite instability (15).

4.7 Lessons from Investigations into COX-1 - Novel Strategies Exploiting the Role of 15-PGDH in Tumourigenesis Hence, it is suggested that molecules that both suppress pro-inflammatory cytokines and induce TGF-ß signalling could act as chemopreventative agents. Such an agent, 2-cyano-3,12-dioxooleana1,9(11)-dien-C28-methyl ester (CDDO-Me), has been shown to suppress colorectal tumourigenesis derived from an inflammatory background (252). In addition to suppressing pro-inflammatory cytokine-induced epithelial proliferation, this molecule also enhanced expression of 15-PGDH in these mice in a TGF-ß dependant mechanism (252). CDDO-Me belongs to a group of molecules

79 known as triterpenoids. It is worth mentioning that common triterpenoids have been shown to have a wide range of functions that could be linked to chemoprevention (257, 258). Hence, it is difficult to suggest whether it is specifically the inhibition of pro-inflammatory cytokines and up-regulation of 15-PGDH that is reducing colorectal cancer risk. Nonetheless, these results suggest that stimulation of 15-PGDH may be a feasible target in the prevention of colorectal cancer and future investigation into more specific inducers of 15-PGDH expression in other models of colorectal tumourigenesis will surely follow in years to come.

Investigations into enhancing 15-PGDH expression have demonstrated potentially novel functions of COX inhibitors. A recent landmark study elegantly described additional mechanisms of PGE2 downregulation for the COX-2 inhibitor apricoxib (259), which has previously been shown to markedly improve colorectal cancer survival in vivo (260). This group investigated this inhibitor in head and neck squamous cell carcinoma (HNSCC), which shares some characteristics with colorectal cancer in that it arises from an epithelial origin and goes through a multi-stage carcinogenesis.

Additionally, COX-2 overexpression has been shown to mediate malignancy (261) and COX-2 inhibition is a promising preventative and therapeutic strategy (262) for this malignancy.

The study by St. John et al demonstrated that in addition to inhibition of COX-2 and PGE2 synthesis, apricoxib also upregulated the PGE2 transporter, PGT, and 15-PGDH (259). With downregulation of 15-PGDH thought to be important in COX-1 mediated tumourigenesis, it may be the case that the activities of apricoxib described by this group indicate that this COX-2 inhibitor may also be pertinent in preventing mechanisms of COX-1 derived tumourigenesis through preventing accumulation of basal PGE2. Of course, it must be remembered that while HNSCC shares characteristics with colorectal cancer, findings in one may not be applicable to the other. Additionally, this study was performed at a stage of tumourigenesis in which COX-2 had become overexpressed and hence the effect of COX-1 derived PGE2 may be negligible. Therefore, it is not clear whether it may have this activity in a background lacking COX-2 overexpression.

80 However, with more and more evidence suggesting a role for COX-1 in colorectal tumourigenesis and concerns remaining over the significant toxicity associated with inhibition of COX-2, addressing the paucity of COX-1 inhibitors may be of great benefit.

4.8 Directions for Further Study - Developing Novel COX-1 Inhibitors While the role for COX-1 in colorectal tumourigenesis is becoming increasingly clear, prospects for prophylactic COX-1 specific NSAID use are hampered by the fact that very few naturally occurring COX-1 selective inhibitors exist. This may be due to the importance of COX-1 in numerous physiological environments. A recent study attempted to address this paucity and in doing so highlighted potential mechanisms that would allow for elucidation of novel COX-1 inhibitors. This group suggested that novel COX-1 inhibitors may be discovered by computational analysis of compound binding specificities (263). Through this analysis Li et al described a novel COX-1 specific inhibitor, 6-C-(E-phenylethyl) Naringenin (6CEPN), which suppressed both the growth of cancer cells without cell contacts and tumour promotion; functions previously shown in this study to be mediated by COX-1 early in tumourigenesis (263). They also demonstrated a remarkable decrease in tumour size of up to 59% compared to untreated mice in xenograft mouse models (263).

This study also highlighted further areas that require further attention. It has classically been established that selective inhibition of COX-1 is responsible for the considerable gastric toxicity associated with the use of some NSAIDs. However, this group reported that 6CEPN caused no gastrointestinal side effects in the mice that were given daily dosages. The exact cause of gastrointestinal toxicity has been cast into doubt before. It has previously been shown that COX-1 knockout mice suffered little gastrointestinal toxicity despite showing significantly reduced PGE2 expression (90) and furthermore a still controversial claim suggests that it is in fact the combination of COX-1 and COX-2 inhibition that causes such side effects (264). Since most of the widely investigated NSAIDs in the field, such as aspirin and sulindac, can inhibit both COX isoforms at sufficient concentrations, it may be the case that such a mechanism has not been appreciated.

81 It is clear therefore, that the investigations into the role of COX-1 in tumourigenesis will be aided by further elucidation of COX-1 selective NSAIDs. This is likely to occur through the use of molecular modelling to screen protein databases for novel inhibitors. Furthermore, since the clinical feasibility of NSAIDs is largely defined by their propensity to cause adverse side-effects, the mechanisms responsible for these must be re-visited, as recent findings may have called into question classically defined mechanisms.

4.9 The Role of COX-1 in Tumourigenesis â&#x20AC;&#x201C; Concluding Statements While numerous roles have been attributed to the COX-2 isoform in tumourigenesis, COX-1 has classically been ignored in the literature. Over the last decade, an increasingly clear role for COX-1 in tumourigenesis has been defined. First suggestions of its role came from studies involving COX-1 knockout mice (90) and expression of COX-2 in colorectal adenomas (91). Later, it was established that COX-1 could directly activate a number of pro-carcinogens epidemiologically linked to colorectal cancer (95). First suggestions of a direct role in tumourigenesis came from studies that suggested COX-1 may play a role in the survival response of intestinal stem cells upon treatment with radiation or a common carcinogen (94, 240). Recently, a mechanism of COX-1-mediated tumourigenesis that elegantly defined a role for aberrant Wnt signalling in inhibiting 15-PGDH was suggested, providing a role for COX-1 derived PGE2 in the early stages of tumourigenesis (241). As a result, 15-PGDH is receiving significant attention, with polymorphisms in this gene potentially mediating resistance to NSAIDs and perhaps pre-disposing to colorectal cancer. Additionally, strategies that induce 15-PGDH expression have provided encouraging results and may yet be a feasible additional strategy alongside NSAID mediated chemoprevention (252, 259).

82

5. Concluding Statements Decades of research are beginning to provide mechanistic explanations for the observations of the effect of NSAIDs on cancer prevention in observational studies and clinical trials. For example, it has long been known that regular use of sulindac results in significant polyp regression (54, 56). Such an activity would rely on the clearance of a population of putative cancer stem cells that feed growing tumours, an activity only recently attributed to COX-2 inhibition (89, 265). Additionally, numerous trials have reported that regular use of aspirin and other NSAIDs reduce the incidence of both colorectal adenomas and colorectal cancer. A finding that could be ascribed to the long-reported and varied effects of COX-derived PGE2 in mediating cancer cell survival. Indeed, exploring this link further, the disparity between the so-called short term, â&#x20AC;&#x2DC;anti-tumourâ&#x20AC;&#x2122; effects and long term, chemopreventative activities associated with regular NSAID use may also be putatively explained by recent studies. While a consensus has largely been reached over the latency associated with NSAID prophylaxis for beneficial chemoprevention, thought to be around 10 years by most, and the suggested mechanisms for this prevention of cancer onset are huge in number, recent studies have suggested shorter period of prophylaxis may also be beneficial. It has been shown that aspirin can reduce both cancer incidence and deaths in a much shorter time frame than previously suggested (62). Further, through the use of aberrant crypt foci (ACF) as trial endpoints, it has been shown that NSAID prophylaxis for just a few months may provide a longer-term benefit (266). The suggested mechanisms for this activity include the inhibition of angiogenesis and metastasis in cases of established tumourigenesis and, as previously discussed, the potential selective clearance of cancer stem cells.

Recent findings that putatively identified a mechanism for COX-1 mediated tumourigenesis (241) may provide an explanation for why COX-2 overexpression, clearly a key mediator of colorectal tumourigenesis, is rarely detected in very early stage adenomas (91) Indeed, this finding may also be applied to explain the partial regression seen by some patients taking regular sulindac (55, 110).

83 Since variations in 15-PGDH expression are thought to determine colorectal cancer risk and potential benefit from NSAID prophylaxis (251, 267, 268), it may be the case that such perturbations in this pathway may contribute to understanding the observations made over the last decades.

Though decades of observational studies and clinical trials have provided encouraging evidence for NSAID-mediated chemoprevention of colorectal cancer, the desire for more efficacious drugs that provide even more protection is natural. However, given the potent toxicity associated with regular NSAID use, the difficulty lies in making these drugs effective without increasing their toxicity even further beyond clinical feasibility. As such, there has been a concerted effort to generate chemicallymodified NSAIDs that retain their activity but obviate the risk of severe adverse effects. One such compound that has received significant attention is nitric-oxide-releasing aspirin (NO-aspirin). This compound has been suggested to have both improved efficacy (269, 270) and safety (271) compared to its native counterpart and its effect on various tumourigeneses is beginning to be elucidated (272, 273). Optimism has been tempered by the as-yet undefined role for nitric-oxide (NO) itself in tumourigenesis. Since NO is inherently genotoxic, yet curiously may have both tumour-promoting and inhibitory activities, the long-term safety of such compounds remains unknown (274).

Other modified NSAIDs have similarly been shown to be more efficacious and markedly safer than native drugs (275-277). Interestingly, these studies reported largely COX-independent mechanisms of chemoprevention, perhaps suggesting that these NSAID derivatives may have additional antineoplastic activities.

The determination of a hazard-to-benefit ratio for individuals potentially allocated to NSAID prophylaxis is an extremely important consideration. The considerable risks associated with regular NSAID use mean that it is not currently a recommended chemopreventative strategy in the general population. Therefore, re-balancing this ratio is of paramount importance. With the questionable safety of long-term NSAID prophylaxis clearly identified, a concerted effort to define benefit from short-term prophylaxis is underway. Randomised trials suggest that short term use of aspirin (62)

84 and sulindac (266) may still provide some degree of prevention of disease. Hence, steps towards a safer regimen of NSAID prophylaxis are already being undertaken.

Furthermore, selection of the appropriate target population that would derive most benefit from NSAID prophylaxis has long been an obstacle to the clinical feasibility of NSAID-mediated chemoprevention. It is natural to suggest that those at high risk of developing colorectal cancer due to pre-disposition syndromes such as FAP and HNPCC will benefit most and though these patients make up a small percentage of the overall cases of colorectal cancer, they have been the subject of recent and continuing trials (28, 108, 109). Further, molecular epidemiology is likely to play a role in identifying patients that will benefit most from NSAID prophylaxis on a personalised basis. Recently, components and metabolites of the prostanoid pathway, which is highly implicated in colorectal tumourigenesis, have been identified as biomarkers that predict both risk of developing colorectal cancer and response to NSAID prophylaxis; it has been suggested that single nucleotide polymorphisms (SNPs) in 15-PGDH and intrinsically low levels of this enzyme may serve as risk factors for colorectal cancer (249, 267). Similarly, markers of high PGE2 metabolism and specific mutations within mitogenic pathways have been identified as predictors of response to NSAID use (278, 279) and it has recently been shown that low levels of 15-PGDH can mediate resistance to particular NSAIDs (267) (see Figure 19). Furthermore, elucidating the genetic basis for predisposition to side effects of NSAID usage will be worth further investigation (59).

A pertinent challenge in this field will be the collation of such biomarkers to the extent that the potential benefits and risks of NSAID prophylaxis can be defined on an individual patient basis. For example, inherently low 15-PGDH expression could be seen as a predictor of benefit from a COX-1 inhibitor, such as aspirin, indomethacin or sulindac (268). Further, high levels of markers of cancer stem cells, such as EphB2, LGR5 and ASCL2, may be predictive of benefit from NSAIDs as an adjuvant therapeutic strategy to prevent recurrence of disease (89, 265, 280).

85

Refs [281], [282], [283]

[278] [284 [285] ] [267], [268]

Figure 19 â&#x20AC;&#x201C; Numerous markers are beginning to be seen as predictors of both colorectal cancer risk and NSAID response.

Variation among the constituents of pathways discussed throughout this dissertation have been linked to either an increased risk of developing colorectal cancer or as predictors of benefit from NSAID prophylaxis. It has been established that aspirin has a greater chemopreventative effect on tumours that overexpress COX-2 (281) and certain polymorphisms in the COX-2 gene have been associated with an increased risk of colorectal cancer and varied efficacy of aspirin to prevent disease (282, 283).

Furthermore, both SNPs and varying levels of expression (indicated by stars) of 15-PGDH have been associated with an increased risk of colorectal cancer (249). It has been suggested that such perturbations in PGE 2 metabolism may be predictive of resistance to or benefit from certain NSAIDs (267, 268). Retrospective analysis of markers of enhanced levels of PGE 2 has also been reported as risk factors for colorectal cancer and markers of a positive response to NSAID prophylaxis (278).

Additionally, the presence of a mutated PI3KCA allele (284) and a wild-type BRAF allele (285) have been associated in epidemiological studies with benefit from use of aspirin.

Furthermore, the benefit of additional disease prevention mediated by NSAIDs will likely play a role in the use of NSAIDs in certain individuals. Since NSAIDs have been linked to the prevention of Alzheimerâ&#x20AC;&#x2122;s disease (35, 286) and aspirin is known to protect against adverse cardiovascular events, it may be the case that individuals at high risk of developing these diseases in addition to cancer may benefit more from NSAID prophylaxis, as potential side effects are inherently less of a deterrent if multiple diseases may be prevented.

86 In conclusion, the field of NSAID-mediated chemoprevention has now reached a stage where sufficient evidence has been accrued to determine its efficacy in certain clinical scenarios and its clinical feasibility on a personalised basis. Though discoveries continue to be made and further complexity will inevitably arise in coming years through the continued elucidation of the complex tumourigenesis of colorectal cancer and the varied roles played by NSAIDs in its prevention, this will allow for continued fine tuning of the hazards and benefits associated with NSAID prophylaxis and therapy.

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