In Canada, colorectal cancer is the second leading cause of cancer death for both men and women. There are many different factors that contribute to the progression and spread of the disease. However, increasing evidence now suggests that the tumor microenvironment plays a paramount role in these processes. CD26 is a multifunctional, cell-surface glycoprotein that has intrinsic enzyme activity, binds adenosine deaminase and interacts with the extracellular matrix. Through its various functions it serves to constrain cancer progression. For example, it is known to cleave CXCL12, the ligand for CXCR4. The CXCL12:CXCR4 axis is normally involved in cancer metastasis by promoting cancer cell migration, invasion and proliferation. Down-regulation of CD26 is observed in certain cancers - this has been shown in vitro to occur in response to certain soluble mediators. The first part of this study looked at the effects of glucose and its metabolic product lactate on CD26 expression in colorectal carcinoma cells. Our study showed that CD26 expression is lower in cancer cells that are grown in low-glucose, high-lactate conditions, which replicates the situation within a tumor. The second part of this study examined the effect of adenosine, a purine nucleoside, on colorectal carcinoma cells and supportive stromal cells - cancer-associated HS675.T fibroblasts (CAFs) and Met-5a mesothelial cells. Adenosine increased the proliferation of CAFs and increased CXCL12 mRNA in both stromal cell lines. It also increased MMP-13 mRNA in stromal cells as well as colorectal cancer cells, suggesting that adenosine may promote progression and metastasis through various mechanisms. The last section focused on the ability of cellular products and 3-dimensional tissue topology to coordinate and affect the behaviour of the different cell populations. Here we show that secretory products from colorectal cancer cells promote CAF proliferation but inhibit mesothelial cell proliferation, and are also able to modulate MMP-13 expression. Finally, certain responses are enhanced in multicellular spheroids. In conclusion, the tumor microenvironment represents a major consideration in the treatment of solid tumors. Our data suggest that various soluble mediators, such as adenosine, may have therapeutic implications in cancer treatment and might represent novel targets for future research.

Loss of genomic stability is associated with a variety of diseases, particularly cancer. Of the many proteins which maintain genomic integrity, two of the most important are MLH1 and MSH2, which participate in DNA mismatch repair. Previous work established derivatives of the CaCo2 human colorectal cancer cell line with siRNA-mediated knockdown of these proteins. When xenografted into mice, tumors with reduced MLH1 or MSH2 expression grew faster than controls. Following growth in vivo, clonal cell lines were established from the tumors and used to examine the effects that knockdown of MSH2 had on other members of the DNA mismatch repair system. Clonal survival following exposure to 5-fluorouracil was also evaluated, and those cells with reduced MLH1 and MSH2 levels were found to be resistant. This study has implications for the importance of knowing the MMR status of a given tumor when deciding on a course of treatment, and of the compounding effects of the loss of one MMR protein on others in the family. / Canadian Cancer Society Research Institute

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The primary treatment for colorectal cancer is surgery. Radiotherapy and chemotherapy, sometimes combined, are also frequently used to diminish recurrence risk. In response to radiation exposure, several cellular signaling cascades are activated to repair DNA breaks, prevent apoptosis and to keep the cells proliferating. Several proteins in the radiation response and cell survival pathways are potential targets to enhance the effects of radiation. The epidermal growth factor receptor (EGFR), which is frequently upregulated in colorectal cancer and exhibits a radiation protective function, is an attractive target for treatment. EGFR is activated by radiation which in turn activates numerous signaling pathways such as the PI3 kinase/AKT cascade, the RAS/RAF/ERK pathway and STAT leading to tumor cell proliferation. EGFR is also believed to interact with proteins in the DNA repair process, such as DNA-PKcs and MRE11. The cytotoxic effect of an affibody molecule (ZEGFR:1907)2, with high affinity to EGFR,  in combination with radiation produced a small, but significant, reduction in survival in a KRAS mutated cell line. However, not in the BRAF mutated cell line. The next step was therefore to target proteins downstream of EGFR such as AKT. There was an interaction between AKT and the DNA repair proteins DNA-PKcs and MRE11 and both AKT1 and AKT2 were involved in the radiation response. The knockout of both AKT isoforms impaired the DNA double strand break rejoining after radiation and suppression of DNA-PKcs increased the radiations sensitivity and decreased the DNA repair further. The AKT isoforms also affected the expression of cancer stem cell markers CD133 and CD44 which are associated with the formation of metastasis as well as radiation and drug resistance. The CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. AKT was also involved in cell migration, cell-adhesion and metabolism. Overall, these results illustrate the complexity in response to radiation and drugs in cells with different mutations and the need for combining inhibitors against several targets such as EGFR, AKT, DNA-PKcs, CD133 or CD44.

The primary aim of this thesis was to conduct a longitudinal study, to investigate the quality of life (QOL) in a large sample of people diagnosed with colorectal cancer (CRC), immediately post-diagnosis and one-year later. Various measures were utilised to capture the multifaceted concept of QOL, including psychological distress, satisfaction with life, positive affect and cancer-related quality of life. The investigation was conducted within Lazarus and Folkman’s stress and coping framework. Specifically, this research aimed to test the utility of the stress and coping framework in the context of CRC, and to explore the role of benefit-finding within the context of the stress and coping model components. One thousand, eight hundred participants (1078 men and 722 women) with a CRC diagnosis duration of 1 to 12 months, completed a telephone interview and written questionnaire, assessing demographics, disease/treatment characteristics, threat appraisal, social support, optimism, coping, benefit-finding and quality of life domains, at approximately five- and 12-months post-diagnosis. To identify CRC-specific coping strategies, the Coping with Colorectal Cancer measure was developed in the initial study. The eight factor structure was confirmed, and the subscales (Positive Perceptual Change; Religion/Spirituality; Rumination; Acceptance; Humour; Palliative; Seeking Social Support; and Lifestyle Reorganisation) found to have reliability and preliminary criterion-related validity within the context of the stress and coping framework. As predicted, regression analyses showed that, after controlling for demographics, disease/treatment characteristics and stress/coping variables, the coping subscales uniquely predicted Time 1 QOL outcomes, with Seeking Social Support continuing to predict Time 2 Positive Affect. In the second study, the benefit-finding domains specific to those with CRC were identified, and relationships with quality of life outcomes assessed, to determine the inclusion of benefit-finding in the path model. Based on completed data from 1757 of the initial 1800 participants, confirmatory factor analyses revealed three domains of benefit-finding: Personal Growth; Interpersonal Growth and Acceptance. As hypothesised, regressions analyses found that benefit-finding domains at Time 1 was associated with Time 1 QOL outcomes, specifically, Positive Affect and Cancer-related Quality of Life (both the aggregate score and its Social/Family, Functional and Colorectal Cancer-specific Well-being subscales). Time 1 Personal Growth also predicted Time 1 Psychological Distress. After controlling for Time 1 Positive Affect, Personal Growth continued to predict Time 2 Positive Affect. The final study drew on the results of the first two studies, which informed the coping strategies and benefit-finding domains to be included in regression analyses initially, and then, structural equation modelling. The final study included 1276 complete data sets of the initial 1800 participants. Parameters of the initial hypothesised model of the stress and coping framework, including relationships with benefit-finding (based on empirical findings) failed to fit the model to the data. After several revisions, the analysis revealed that the final model fit the data, where stress, coping and benefit-finding accounted for 63% of the variance in Time 1 QOL. The model showed that threat appraisal, coping resources, avoidant coping and benefit-finding directly impacted on Time 1 QOL, while threat appraisal, social support and approach coping directly impacted on benefit-finding. In this study, the approach coping strategies included in the path model could also be conceptualised as meaning-based coping strategies, as they appeared to facilitate a meaning-making process. However, benefit-finding, which some researchers have suggested is also a meaning-based coping strategy, had differential relationships with stress, coping and outcome variables, compared with the approach coping strategies. These results indicate that benefit-finding is an empirically distinct construct in the context of CRC. Finally, in this study, the impact of stress, coping and benefit-finding on Time 2 QOL, was indirect, being mediated by Time 1 QOL outcomes. The accumulated findings of these three studies have extended the cancer coping and benefit-finding research by revealing new relations between stress, coping and benefit-finding and QOL in a mixed-gender, older population with CRC. There are implications for measurement of, and theory building around benefit-finding. Finally, these studies inform the development of clinical interventions to enhance the quality of life in the short- and longer-term for individuals diagnosed with CRC.

The primary aim of this thesis was to conduct a longitudinal study, to investigate the quality of life (QOL) in a large sample of people diagnosed with colorectal cancer (CRC), immediately post-diagnosis and one-year later. Various measures were utilised to capture the multifaceted concept of QOL, including psychological distress, satisfaction with life, positive affect and cancer-related quality of life. The investigation was conducted within Lazarus and Folkman’s stress and coping framework. Specifically, this research aimed to test the utility of the stress and coping framework in the context of CRC, and to explore the role of benefit-finding within the context of the stress and coping model components. One thousand, eight hundred participants (1078 men and 722 women) with a CRC diagnosis duration of 1 to 12 months, completed a telephone interview and written questionnaire, assessing demographics, disease/treatment characteristics, threat appraisal, social support, optimism, coping, benefit-finding and quality of life domains, at approximately five- and 12-months post-diagnosis. To identify CRC-specific coping strategies, the Coping with Colorectal Cancer measure was developed in the initial study. The eight factor structure was confirmed, and the subscales (Positive Perceptual Change; Religion/Spirituality; Rumination; Acceptance; Humour; Palliative; Seeking Social Support; and Lifestyle Reorganisation) found to have reliability and preliminary criterion-related validity within the context of the stress and coping framework. As predicted, regression analyses showed that, after controlling for demographics, disease/treatment characteristics and stress/coping variables, the coping subscales uniquely predicted Time 1 QOL outcomes, with Seeking Social Support continuing to predict Time 2 Positive Affect. In the second study, the benefit-finding domains specific to those with CRC were identified, and relationships with quality of life outcomes assessed, to determine the inclusion of benefit-finding in the path model. Based on completed data from 1757 of the initial 1800 participants, confirmatory factor analyses revealed three domains of benefit-finding: Personal Growth; Interpersonal Growth and Acceptance. As hypothesised, regressions analyses found that benefit-finding domains at Time 1 was associated with Time 1 QOL outcomes, specifically, Positive Affect and Cancer-related Quality of Life (both the aggregate score and its Social/Family, Functional and Colorectal Cancer-specific Well-being subscales). Time 1 Personal Growth also predicted Time 1 Psychological Distress. After controlling for Time 1 Positive Affect, Personal Growth continued to predict Time 2 Positive Affect. The final study drew on the results of the first two studies, which informed the coping strategies and benefit-finding domains to be included in regression analyses initially, and then, structural equation modelling. The final study included 1276 complete data sets of the initial 1800 participants. Parameters of the initial hypothesised model of the stress and coping framework, including relationships with benefit-finding (based on empirical findings) failed to fit the model to the data. After several revisions, the analysis revealed that the final model fit the data, where stress, coping and benefit-finding accounted for 63% of the variance in Time 1 QOL. The model showed that threat appraisal, coping resources, avoidant coping and benefit-finding directly impacted on Time 1 QOL, while threat appraisal, social support and approach coping directly impacted on benefit-finding. In this study, the approach coping strategies included in the path model could also be conceptualised as meaning-based coping strategies, as they appeared to facilitate a meaning-making process. However, benefit-finding, which some researchers have suggested is also a meaning-based coping strategy, had differential relationships with stress, coping and outcome variables, compared with the approach coping strategies. These results indicate that benefit-finding is an empirically distinct construct in the context of CRC. Finally, in this study, the impact of stress, coping and benefit-finding on Time 2 QOL, was indirect, being mediated by Time 1 QOL outcomes. The accumulated findings of these three studies have extended the cancer coping and benefit-finding research by revealing new relations between stress, coping and benefit-finding and QOL in a mixed-gender, older population with CRC. There are implications for measurement of, and theory building around benefit-finding. Finally, these studies inform the development of clinical interventions to enhance the quality of life in the short- and longer-term for individuals diagnosed with CRC.

The aim of this research was to assemble sufficient experimental evidence about candidate gene transcript expression changes between non-neoplastic and neo- plastic colorectal tissues to justify future assay development involving promis- ing leads. To achieve this aim, this thesis explores the hypothesis that gene expression-based biomarkers can be used to accurately discriminate colorectal neoplastic tissues from non-neoplastic controls. This hypothesis was tested by first analysing multiple, large, quality controlled data sets comprising gene expression measurements across colorectal phenotypes to discover potential biomarkers. Candidate biomarkers were then subjected to validation testing using a custom-design oligonucleotide microarray applied to independently derived clinical specimens. A number of novel conclusions are reached based on these data. The most important conclusion is that a defined subset of genes expressed in the colorectal mucosa are reliably differentially ex- pressed in neoplastic tissues. In particular, the apparently high prediction accu- racy achieved for single gene transcripts to discriminate hundreds of neoplastic and non-neoplastic tissues provides compelling evidence that the resulting can- didate genes are worthy of further biomarker research. In addition to addressing the central hypothesis, additional contributions are made to the field of colorectal neoplasia gene expression profiling. These contributions include: The first systematic analysis of gene expression in non-diseased tissues along the colorectum To better understand the range of gene expression in non-diseased tissues, RNA extracts taken from along the longitudinal axis of the large intestine were studied. The development of quality control methodologies for high dimen- sional gene expression data Complex data collection platforms such as oligonucleotide microarrays introduce the potential for unrecognized confound- ing variables. The exploration of quality control parameters across five hundred microarray experiments provided insights about quality control techniques. The design of a custom microrray comprised of oligonucleotide probe- sets hybridising to RNA transcripts differentially expressed in neo- plastic colorectal specimens A custom design oligonucleotide microarray was designed and tested combining the results of multiple biomarker discovery projects. Introduction of a method to filter differentially expressed genes dur- ing discovery that may improve validation efficiencies of biomarker discovery based on gene expression measurements Differential expression discovery research is typically focused only on quantitative changes in transcript concentration between phenotype contrasts. This work introduces a method for generating hypotheses related to transcripts which may be quali- tatively “switched-on” between phenotypes. Identification of mRNA transcripts which are differentially expressed between colorectal adenomas and colorectal cancer tissues Transcripts differentially expressed between adenomatous and cancerous RNA extracts were discovered and then tested in independent tissues. In conclusion, these results confirm the hypothesis that gene expression profiling can discriminate colorectal neoplasia (including adenomas) from non-neoplastic controls. These results also establish a foundation for an ongoing biomarker development program.

Together, colorectal (CRC) and bladder cancer (BlCa) are responsible for a large percentage of cancer related morbidity and mortality in Western society. A dramatic reduction in patient survival occurs as these cancers progress towards invasive and metastatic disease, from a five year survival rate of about 90% for localised disease to approximately 5-10% for advanced disease involving distant metastasis. A greater understanding of disease progression will lead to enhanced screening, diagnostic and treatment strategies, in turn providing an improved prognosis for the patient. The purpose of this study was to expand the current molecular knowledge of CRC and BlCa by elucidating the role of Mxi1 mutations and MTSS1 expression in CRC and BlCa respectively, and to examine the diagnostic potential of these genes. The Mxi1 coding region for 41 tumours, collected by the South Western Sydney Colorectal Cancer Tumour Bank from 2000-2001, was screened for mutations using Dideoxy Fingerprinting (ddF) and sequencing. Sequence alterations were detected in 34% of tumours. Three different polymorphisms and three mutations were detected. One mutation could possibly affect the tumour suppressor function of Mxi1. The presence of a gene mutation did not correlate to any clinical characteristics and is therefore not a suitable diagnostic marker. Microsatellite instability (MSI) status however, significantly correlated with tumour grade. Expression levels of MTSS1 and an associated gene, MTSS2, were examined in 16 BlCa cell lines, 9 clonally-derived BlCa sublines, and 30 transitional cell carcinomas (TCCs) collected by the Heinrich-Heine University from 1993-2000. Variable gene expression was observed in BlCa cell lines and tumour samples. No significant correlation of MTSS expression and invasive ability was observed for the cell lines or tumour samples. Further studies eliminated promoter methylation and p53 functional status as mechanisms involved in MTSS1 and MTSS2 down-regulation. Functional studies performed on stable MTSS1-expressing BlCa lines found that although migration was increased, cells displayed reduced anchorage-independent growth. The invasive ability of these cells was unchanged confirming that expression does not correlate with invasive ability. These data support the role of MTSS1 as a tumour suppressor and not as a metastasis suppressor gene. Although MTSS1 may not be useful in predicting more invasive disease, its role as a tumour suppressor in cancer may be useful.