Colorectal cancer (CRC) is common in Ontario. This study described the processes of care following CRC resection, and identified CRC relapse from administrative data. Methods: CRC patients aged 18-80 from 1996-2001 with a colorectal resection were identified from the Ontario Cancer Registry. Linked discharge abstracts and physician billings were examined for physician visits, body imaging and endoscopy over the 5 year follow-up period. Administrative codes suggesting disease relapse were compared with patient charts. Results: Overall, 12,804 patients were identified and 8,804 had no evidence of relapse. Most (96.2%) patients had general practitioner follow-up, while 49.3% had medical oncology and 80.4% had general surgery follow-up. Greater than 90% of patients received endoscopy, while only 68.7% of patients received body imaging. Detecting disease relapse was 87.5% sensitive and 93.0% specific. Conclusions: There is potential for improving post-resectional follow-up in CRC patients. It is possible to detect relapse through administrative databases.

The Proximity Ligation Assay (PLA) is a relatively new method which utilizes the strength of both immunoassays and DNA detection. PLA has the capacity of high multiplexing due to the high specificity achieved with both dual protein-binding and dual primer binding during detection with Real-Time PCR. We developed a multiplexed PLA protocol that can measure 28 biomarkers in human EDTA plasma. The method was tested on 46 individuals diagnosed with colorectal cancer and 48 age matched healthy controls. The results are very promising as we re-discover the most well-known biomarkers for colorectal cancer and also find some potential new markers (significance tested with students T-test with p<0.05). Further improvements of the protocol are needed to decrease the variation.

Background and ObjectiveColorectal cancer (CRC) remains the second deadliest cancer in the United States. Several screening methods exist; however, detection of small polyps remains a challenge. Optical coherence tomography (OCT) has been demonstrated to be capable of detecting lesions as small as 1mm in the mouse colon, but detection is based on measuring a doubling of the mucosa thickness. The colon microvasculature may be an attractive biomarker of early tumor development because tumor vessels are characterized by irregular structure and dysfunction. Our goal was to develop an endoscopic method of detecting and segmenting colon vessels using Doppler OCT to enable future studies for improving early detection and development of novel chemopreventive agents. MethodWe conducted in vivo colon imaging in an azoxymethane (AOM)-treated mouse model of colorectal cancer using a miniature endoscope and a swept-source OCT system at 1,040nm with a 16kHz sweep rate. We applied the Kasai autocorrelation algorithm to laterally oversampled OCT B-scans to resolve vascular flow in the mucosa and submucosa. Vessels were segmented by applying a series of image processing steps: (i) intensity thresholding; (ii) two-dimensional matched filtering; and (iii) histogram segmentation. ResultsWe observed differences in the vessels sizes and spatial distribution in a mature adenoma compared to surrounding undiseased tissue and compared the results with histology. We also imaged flow in four young mice (two AOM-treated and two control) showing no significant differences, which is expected so early after carcinogen exposure. We also present flow images of adenoma in a living mouse and a euthanized mouse to demonstrate that no flow is detected after euthanasia. ConclusionWe present, to the best of our knowledge, the first Doppler OCT images of in vivo mouse colon collected with a fiber-based endoscope. We also describe a fast and robust image processing method for segmenting vessels in the colon. These results suggest that Doppler OCT is a promising imaging modality for vascular imaging in the colon that requires no exogenous contrast agents.

Introduction The adjuvant treatment of colorectal cancer (CRC) has seen little improvement in terms of mortality of the disease in the last 40 years. There has been a resurgence in research into the use of monoclonal antibodies in the treatment of CRC. Carcinoembryonic antigen (CEA) is a useful target in cancer immunotherapy. The distribution of CEA in CRC differs from that in normal colorectal tissue. In normal colorectal tissue CEA is found only on the luminal surface of the cell which is inaccessible to intravenous antibody, whereas in CRC, CEA is found on all borders of the cell membrane and so becomes accessible to intravenous antibody. However, anti-CEA antibodies are prone to sequestration by circulating CEA. The anti-CEA antibody, PR1A3, binds only membrane-bound CEA and thus is able to overcome this problem. The aim of my research was to assess whether PR1A3 is suitable to be considered as a therapeutic agent in the treatment of CRC and what its mechanism of action might be. Methods The level of expression of CEA on a panel of cell lines was determined under different conditions using a solid-phase ELISA and FACS analysis. Humanized PR1A3 (hPR1A3) was assessed in a variety of in vitro cytotoxicity assays with colorectal cell lines expressing varying levels of CEA, using peripheral blood mononuclear cells and purified natural killer cells as sources of effector cells. The mechanism of action of PR1A3 was investigated by modifying the Fc fragment of the antibody and using antibodies to block the FcIIIA receptor on the effector cells. PR1A3 was also investigated in combination with a humanised A33 antibody. Results A panel of colorectal cell lines was found to have a range of CEA expression which could be upregulated in certain cell lines by growing the cell line beyond confluence and by treatment with the chemotherapeutic agent, 5-fluorouracil. The in vitro assays demonstrated hPR1A3 antibody-dependent and CEA-specific killing of tumour cell lines by human PBMC. The effect increased with increasing concentration of antibody and was lost by using the parent murine IgG1 PR1A3. Using 50&mu;g/ml hPR1A3, tumour cell lysis was increased by more than 3-fold above spontaneous killing (p < 0.001) in a high CEA-expressing cell line. Both antibody-dependent and antibody-independent (spontaneous) killing was blocked by using whole antibody to the Fc-&gamma;IIIA receptor, although the spontaneous killing was restored when a F(ab')2 was used instead of whole antibody. hPR1A3 and the A33 antibody showed potential synergy when used in combination against a high-CEA and a moderate-A33 expressing cell line. Conclusion The monoclonal antibody hPR1A3 causes CEA-specific lysis of human colorectal cancer-derived cell lines in the presence of human PBMCs. This lysis is dependent on the dose of the antibody, requires a compatible Fc-receptor and is inhibited by blockade of the FcγIIIA receptor. These findings show that hPR1A3 can kill tumour cells by antibody-mediated cellular cytotoxicity (ADCC) and implicate NK cells as a major contributor to this effect. The results support the development of hPR1A3 for therapy of colorectal cancer.

BACKGROUND: Radiation exposure to the abdominal region causes intestinal toxicity and is also capable of inducing colorectal cancers (CRC). Genotype-phenotype studies provide some evidence explaining the variation in familial adenomatous polyposis (FAP) patients caused by modifiers of adenomatous polyposis coli (APC). This study aims to extend our understanding of irradiation-induced modifiers of ApcMin/+ mice and CRC. METHODS: By using a pre-existing backcross between recombinant inbred line of ApcMin/+ mice to the irradiation sensitive inbred BALB/c mouse, we obtained panels of 2Gy-irradiated and sham-irradiated N2 ApcMin/+ mice for genotyping with a genome-wide panel of microsatellites markers. Using the number of adenomas in different intestinal segments to represent polyp multiplicity, we carried out a genome wide quantitative trait loci (QTL) scan followed by statistical epistasis modelling and bioinformatics analysis. RESULTS: We identified five significant QTLs responsible for radiation induced tumour multiplicity in the upper small intestine defined as Mom (Modifier of Min) radiation-induced polyposis (Mrip1-5) on chromosome 2 (LOD 2.8, p = 0.0003), two regions within chromosome 5 (LOD 5.2, p=0.00001, 6.2, p=0.00001) and two regions within chromosome 16 (LOD 4.1, p=4x10-5 and 4.8, p=0.00001). Suggestive QTLs were found for sham-irradiated mice on chromosomes 3, 6 and 13 (LOD 1.7, 1.5 and 2.0 respectively; p,0.005). Two significant QTLs were detected in the 2large intestine on chromosome 2 and 7 (LOD 2.7, p=1.2x10-3 and 2.2, p=1.2x10-3, 12 respectively). Using statistical epistasis modelling and logical selection of target genes though in silico sequence based on BALB/c specific non-synonymous polymorphisms which are predicted deleterious we selected target genes and further eliminated genes by sequencing and mRNA expression. CONCLUSIONS: Our study locates the QTL regions responsible for increased radiation-induced intestinal tumorigenesis in ApcMin/+ mice and identifies candidate genes with predicted functional polymorphisms that are involved in spindle checkpoint and chromosomal stability (Bub1b, Bub1r, and Casc5), Wnt pathway (Tiam1, Rac1), DNA repair (Recc1 and Prkdc) and inflammation (Duox2, Itgb2l and Cxcl5).

Background: Colorectal cancer is the result of dysregulation within classic regulatory pathways in epithelial stem-cell(s): the precursors giving rise to all other intestinal lineages. The resulting cancer stem-cell (CSC) generates tumours utilising its innate properties, e.g. self-renewal and lineage plasticity. CSCs appear to persist within a tumour as a distinct subtype responsible for local recurrence/metastasis. Therefore, therapies targeting colorectal CSCs may lead to improved cancer-specific outcome measures. The PROM-1/CD/AC133 cell surface marker has been associated with colorectal CSCs and its expression is reported as an independent negative prognostic marker. Therefore, this thesis sought to investigate the role of PROM-1/CD/AC133 in colorectal cancer. Methods: Tissue-culture, RT-qPCR, IHC, Western blotting, siRNA, PCR-array and FACS analyses were used to quantify and profile mRNA/protein expression patterns. Results: PROM-1/CD/AC133 was widely expressed in patient-matched colorectal tumour, adjacent normal epithelium, vascular invasion, lymph node metastases with significantly decreased expression in liver metastases. Furthermore, PROM- 1/CD/AC133 expression was not found to enrich colorectal cancer cell line populations for additional stem cell phenotypes (expression of ABCB1/ABCG2/BMIJ Murphy 1/CD44/LGR5/MSI-1). The data confirm the presence of alternative splice variants of PROM-1, and show that transcripts specifying PDZ binding predominate in colorectal cancer cell lines. Concomitantly, siPROM-1 was shown to modulate the expression of several key transcripts in colorectal tumourigenesis as well as regulate signal transduction pathways including the central cancer, colorectal cancer, NF-kB and p53 signalling cascades. Conclusions: PROM-1/CD/AC133 does not identify rare colorectal cancer cells responsible for tumourigenesis. However, it is associated with the regulation of signalling networks associated with cell growth, differentiation and apoptosis suggesting a potential role for this marker in colorectal tumourigenesis. The identification of specific target genes and signalling pathways in this thesis provides a springboard for further investigations into the functional role of this marker in colorectal cancer, with the potential for better treatments for this disease.

Introduction: Van Gogh-Like 2 (VANGL2) is a scaffolding planar cell polarity protein involved in non-canonical Wnt signalling. It has been shown to have crucial roles in regulating epithelial development and homeostasis. Moreover, VANGL2 has been implicated in human cancers, with increased expression and copy number amplification seen in several cancer contexts. Many related components within this pathway have also been linked to cancer development, with VANGL2 expression known to regulate factors involved in cell migration and extracellular matrix (ECM) remodelling in cell lines. These cellular processes tend to be erroneously activated in cancer. VANGL2 is known to inhibit the classical driver pathway of colorectal cancer (CRC), canonical, or β- catenin dependant, Wnt signalling, in CRC cell lines. The aim of this thesis is to determine the expression of VANGL2 in CRC, and to investigate how VANGL2 may act to regulate intestinal homeostasis and disease. Methods: Transcriptional verification of VANGL2 expression in the mouse intestine was carried out by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and transcripts localised within the murine colon using RNA-In Situ Hybridisation (RNAISH). Expression and localisation of the VANGL2 protein and related non-canonical Wnt signalling components was confirmed using immuno-histochemistry (IHC). Furthermore, using a combination of human Tissue Micro-Array (TMA), transcriptional data and genomic data, we determined an association between VANGL2 on tumour grade and disease-free survival. To functionally validate the effects of VANGL2 on colorectal biology, we used a model in which VANGL2 is selectively deleted from the colonic epithelium using Villin-CreERT Vangl2flox mouse lines. Using a combination of molecular biology methods, we identified the ECM as differentially regulated following VANGL2 modulation. To test the role of VANGL2 in colorectal cancer, we used a murine colorectal cancer model in which adenomatous polyposis coli (APC) is deleted from colonic epithelium resulting in the formation of cancer concurrently with deletion of Vangl2. We evaluated survival of these mice as well as tumour number and size. Tumour tissue was analysed using IHC, qRT-PCR and 3-Dimensional organoid culture. Results: Within this thesis I have illustrated that the murine colonic epithelium expresses Vangl2, and other components known to interact with VANGL2 including Vangl1, Wnt5A, and Protein Tyrosine Kinase 7 (Ptk7). I have also shown that VANGL2 is expressed within the human colonic epithelium. I go on to show that 9.2% of human CRC possesses VANGL2 transcriptional alterations which correlates with a worsened disease-free survival (DFS) rate among patients. Using IHC, I also show that higher grade CRC is associated with increased VANGL2 expression. In our murine cancer model, mice with single or dual-copy loss of VANGL2 were found to have a reduced number of colonic tumours, while maintaining similar tumour size. Investigations to identify how VANGL2 may have control of tumour initiation were carried out focussing on the ECM. I found that, contrary to what I have discovered in the healthy murine colon, tumours from VANGL2-deficient mice had increased transcription of the ECM markers Secreted protein acidic and rich in cysteine (Sparc) and Decorin (Dcn), as well as increased expression of the ECM regulators Matrix Metallopeptidase 9 (Mmp9) and Tissue Inhibitor of Metalloproteinases 1 (Timp1). Changes in the ECM was also seen at the protein level, with increases in staining for the ECM components Col1 (Collagen, type I), and Laminin in VANGL2-deficient tissue. The ECM modulator Connective Tissue Growth Factor (Ctgf), is implicated in multiple cancers including CRC and is increased within VANGL2-deficient tumours at both the transcript and protein level, implicating Ctgf in increasing the ECM of these tumours.

Large bowel cancer, or colorectal cancer (CRC) is the third most common cause of cancer worldwide and the fourth biggest cause of cancer mortality. Twin studies have shown that the heritable contribution is ~35%, with ~5% of cases due to rare, high-penetrance mutations. In the last decade, the use of genome-wide association studies on large, well-characterised case-control cohorts of CRC has facilitated the identification of over 25 common genetic variants that carry with them an increased predisposition to colorectal cancer, invoking the common-disease common variant paradigm. As almost all of these variants lie within non-coding regions, the underlying causal mechanism is to-date poorly understood for the majority of these loci, and it is thought that they mediate risk by influencing gene expression levels. To test this hypothesis, an agnostic approach that utilises expression quantitative trait loci (eQTL) analysis was first carried on 115 normal colorectal mucosa samples and 59 peripheral blood mononuclear cells (PBMC). As these heritable variation on gene expression are likely to be subtle, there is a strong emphasis on the technical methodology to minimise experimentally-induced non-biological variations, including the extraction of high-quality RNA from primary tissue, the selection and validation of reference genes for normalisation of gene expression quantification, as well as internal validation of the samples and data processing. Thereafter, the association between the 25 CRC risk variants and the expression of their cis-genes were examined systematically, demonstrating that ten of these variants are also tissue-specific eQTLs. This intermediate phenotype strongly suggests that they confer risk, at least in part, by modifying regulatory mechanisms. One of the best eQTL associations (Xp22.2) is investigated in further detail to reveal a novel indel polymorphism (Indel24) at the distal promoter region of target gene SHROOM2 that influenced both transcript abundance and CRC risk more than the original tagging SNP. Functional verification with gene reporter assays indicated that Indel24 displays differential allelic control over transcriptional activity. Further in silico analysis and mutations to the reporter gene constructs provided evidence that Indel24 modulates transcription by modifying the spacing between CCAAT motifs and the consequent binding affinity of NF-Y transcription factor. siRNA depletion of NF-Y was associated with a reduction in transcriptional activity of the Indel24 gene construct as well as endogenous SHROOM2, which is strongly supportive of the interaction between Indel24 and NF-Y in the transcriptional activation of SHROOM2. Preliminary evidence is suggestive of SHROOM2 being expressed at the top of the intestinal epithelial crypt and playing a role in cell cycle regulation. Hypothesis-driven approaches can also be of utility in demonstrating functionality of CRC risk variants, complementing the hypothesis-free approach of eQTL analysis. Guided by a recently discovered gene-environment interaction between the 16q22.1 risk variant and circulating vitamin D levels, the influence of the rs9929218 SNP on CDH1 gene expression was examined, in relation to the expression of putative regulatory genes derived from in silico analysis and studies of other target genes. Although there was no direct association between rs9929218 and CDH1 expression, there were multiple two-way interactions that were together suggestive of rs9929218 influencing the VDR/FOXO4 regulation of CDH1. This provides functional support for the mechanism underlying the epidemiological observation of the gene-environment interaction between 16q22.1 and vitamin D, and demonstrates a candidate-based approach in deciphering the link between genetic locus and CRC susceptibility. In summary, the research presented in this thesis has validated the experimental rationale of utilising expression studies of normal colorectal mucosa to hone in on the molecular mechanisms and susceptibility genes underlying the association between common genetic variation and CRC risk.

Colorectal cancer (CRC) is the third most common cancer globally with around 1.3 million cases diagnosed annually. In cases of inherited CRC where none of the rare, high-risk mutations associated with familial syndromes are observed it has been theorised that the heritable risk is due to common, low-risk variants in the genome. Identifying these variants of modest or small effect size has become possible due to the use of large genome-wide association studies (GWAS). Through a meta-analysis of five previous GWAS, Dunlop et al. identified three novel susceptibility loci at 6p21, 11q13.4 and Xp22.2. The aims of this study were to further characterise the Xp22.2 risk locus and investigate the function of the putative risk gene SHROOM2. The variant originally identified in the Dunlop et al. study as showing an association with CRC risk rs5934683 is located between SHROOM2 and GPR143. Genome–wide expression analysis has shown that the variant is an eQTL (expression quantitative locus), affecting expression of SHROOM2, but not GPR143. An important caveat to this analysis was that the commercial array used to measure gene expression does not detect all predicted GPR143 transcripts. Hence it was important to understand whether GPR143 might be involved at the locus and whether there was altered expression in normal colonic mucosa. I have analysed the expression of GPR143 and shown that it is poorly expressed in normal mucosa and that expression of the alternative transcripts is rare. This provided further evidence for the gene of interest at Xp22.2 being SHROOM2 and thus became the focus for further investigation. In order to understand SHROOM2 function a knockout mouse was generated allowing studies of gene function beyond the previously used in vitro systems. Embryonic stem cells containing a Shroom2 knockout first allele were obtained from the International Knockout Mouse Consortium and a novel mouse line established. This mouse line was found to be an incomplete knockout and a Cre recombination strategy was employed to remove the critical exon and create a true null allele for Shroom2. This model was validated as being a true knockout of Shroom2 at both the RNA and protein level and the model subjected to initial phenotyping focusing on tissues where the gene has previously been identified as expressed. To investigate the role of Shroom2 as a CRC susceptibility gene preliminary data has been gathered from crosses to the ApcMin/+ CRC model, and analysis of the intestines of the Shroom2KO line has been undertaken. Two spontaneously occurring anorectal adenomas have been identified in Shroom2 null mice, and an additional mid-colonic polyp phenotype identified when crossed onto the ApcMin/+ background. Additionally, embryonic fibroblasts have been used in growth and wound healing assays to determine what effect total loss of Shroom2 has at a cellular level. Proteomics analysis to identify significantly altered pathways associated with Shroom2 loss has also been carried out and has highlighted a number of interesting targets for further investigation. In summary, a novel Shroom2 knockout mouse model has been developed to investigate the CRC susceptibility locus identified at Xp22.2. Preliminary data from this mouse model appears to confirm SHROOM2 as having a role in tumour development in the large intestine.

Overwhelming evidence indicates that aspirin (ASA) and related non-steroidal anti-inflammatory drugs (NSAIDs) have anti-tumour activity against colorectal cancer (CRC). Although the underlying mechanisms have yet to be fully elucidated, the host laboratory have shown that nucleolar sequestration of the NF-κB component RelA is critical. In the course of these studies, it was noted that alongside effects on the NF- κB pathway, ASA has a profound effect on nucleoli, including a dramatic increase in nucleolar size. These data were particularly interesting as, in addition to its role in ribosome biogenesis, the nucleolus is known to act as a stress sensor and play a key role in the regulation of cell growth and apoptosis. Indeed, this organelle has been identified as a potential target for anti-tumour agents. However, how stress causes changes to nucleolar function, and how these are translated into changes in cell phenotype, remain unclear. Therefore, the aim of my thesis was to fully characterise ASA effects on nucleoli and to determine whether these effects contribute to the anti-tumour activity of this agent. I found that ASA induced an atypical form of nucleolar stress that was associated with enlargement of the organelle, relocalisation of nucleolar markers to the periphery, depletion of the critical component of the Pol I transcription factor complex, TIF-IA, and inhibition of rRNA transcription. These effects were independent of the p38 and JNK2 MAP kinase pathways. However, they were mimicked by inhibition of CDK4, which had previously been shown to lie upstream of ASA effects on the NF-κB pathway. These data describe a novel mechanism by which ASA, and CDK4 inhibition, may inhibit the growth of colon cancer cells. In addition to this candidate approach, I used Stable Isotope Labelling by Amino acids in Cell culture (SILAC) based quantitative proteomics to obtain a global overview of ASA effects on nucleoli of colon cancer cells. Firstly, a protocol was successfully developed to isolate pure nucleoli from SW480 CRC cell lines. This protocol was then applied to SILAC labelled cells treated with ASA for three time-points (0, 6, 10 h). In collaboration with R.T Hay and M. Tatham (University of Dundee), proteomic analysis was then carried out by tandem-mass spectrometry. These data confirmed that ASA has a significant effect on the nucleolar proteome. They also revealed that ASA induces a distinct type of nucleolar stress that is associated with the accumulation of chaperones, translational regulators and members of the ubiquitin-proteasome system (UPS) in this organelle. These data were reminiscent of studies previously published on the effect of proteasome inhibition on nucleoli. I therefore used SILAC-based proteomics to compare ASA effects on nucleoli to those induced by the proteasome inhibitor, MG132. I found that similar sub-groups of proteins accumulate in nucleoli in response to both agents and that ASA induced proteotoxic stress in a similar manner to MG132. Fluorescence correlation spectroscopy in collaboration with R. Duncan and K. Martin (Heriot-Watt University) demonstrated the relative reduction in mobility of nucleolar DsRed-RelA, indicating that, similar to MG132, ASA induces formation of nucleolar aggresomes. Mechanistic studies suggested that blocking ASA-mediated proteotoxic stress blocked the apoptotic effects of the agent. Taken together, these data define a distinct type of nucleolar stress that may be involved in the cells response to proteotoxic stress and be required for the anti-tumour activity of ASA.