Colorectal cancers (CRCs) rely heavily on host-derived structures to support tumour development and maintain growth. The tumour microenvironment (TME) includes contributions from underlying vascular endothelial cells to maintain nutrient and oxygen availability, as well as a delicate interplay with effector and suppressor immune cells to ensure effective immune evasion. Despite previous attempts to target these TME components through direct, individual therapeutic interventions targeting pro-angiogenic signaling and blocking immune suppression, in many cases the CRC TME remains supportive of tumour growth. Here, I have identified a loci-specific Autism Spectrum Disorder-linked 16p11.2 microdeletion, that demonstrates a negative impact on tumour growth. Initially, I show that a constitutive 16p11.2 deficiency (16p11.2df) alters tumour growth, although has no impact on tumourigenesis. Moreover, I demonstrate that a 16p11.2df in host- derived structures of the TME but not tumour cells is sufficient to slow tumour growth. Further, I demonstrate that vascular networks are altered in tumours derived from a 16p11.2df TME. While I present systemic alterations to the immunological landscape of 16p11.2df individuals at the transcriptional level, there is no detectable alteration in cytotoxic immune cell infiltration into 16p11.2df-derived tumours despite increased expression of T cell activating cytokines. In addition to altered growth characteristics, tumours derived from 16p11.2df mice show an enrichment in apoptosis of tumour cells, despite no change in proliferation. These findings suggest the potential for a 16p11.2df-mediated induction of tumour dormancy, through which volumetric tumour growth is slowed. Together, this study demonstrates the need for further investigation of the 16p11.2 microdeletion as a critical regulator of tumour growth.

Background and PurposeGlobally and in the United States (US), colorectal cancer (CRC) is the second leading cause of cancer-related death, following lung cancer. In addition, there are established racial disparities in incidence and mortality for this disease, where ethnic minority groups have higher incidence and mortality rates. Blacks currently have the second-highest rates of CRC incidence and mortality, are diagnosed at more advanced stages, and have the lowest 5-year survival rates of all racial groups. Multiple influences impact this disparity including area- and individual-level factors. Area-level factors, encompassing social determinants of health (e.g. area-level poverty, housing characteristics, etc.), play a role in disease etiology and outcomes. In addition, timely CRC screening (CRCS) reduces CRC incidence and mortality; however, screening patterns, globally and in the US, are not optimal and differ by race, with ethnic minority groups having low CRCS adherence compared to non-Hispanic whites. Differences in CRCS behaviors and outcomes have been noted for Blacks, a term used to describe, for example, a heterogeneous racial group comprised of US-born Blacks and immigrants from Africa and the Caribbean. While CRCS barriers are well documented for the general population, CRCS barriers are less understood for Blacks and very little is known about CRCS habits and CRCS barriers within this heterogeneous racial group, with limited research including Caribbean immigrants and no known research including African-born immigrants. This dissertation uses a mixed-methods approach to describe CRC incidence, advanced stage at diagnosis, and mortality, CRCS behaviors, and CRCS barriers within the heterogeneous Black population in Philadelphia County, Pennsylvania. Aim 1. Assess colorectal cancer incidence, advanced stage at diagnosis, and colorectal cancer mortality, overall and among individuals who identify as Black/African American, and contextual disparities in Philadelphia County utilizing data from the Pennsylvania State Cancer Registry (2010–2016) and relevant US Census and American Community Survey data. Aim 2. Determine colorectal cancer screening adherence for Cancer Prevention Project of Philadelphia (CAP3) participants who self-identify as Black. Aim 3: Conduct gender-specific focus groups to elucidate the principal barriers to colorectal cancer screening adherence within an average-risk group of adults, ages 45–75, who self-identify as Black or African American, in an urban population. Methods Aim 1. Using an ecological design, descriptive, geographic spatial clustering and hierarchical logistic regression analyses were done to describe CRC incidence, advanced stage at diagnosis, and colorectal cancer-specific mortality in Philadelphia County at the individual- and area-level. CRC incidence, stage at diagnosis, and mortality data for histologically confirmed CRC cases were obtained from the Pennsylvania Cancer Registry from 2010 to 2016, with mortality data including deaths through 2020. Area-level data were retrieved from the US Census Bureau, American Community Survey, etc. Individual- and area-level descriptive characteristics were calculated for all CRC incident cases, cases diagnosed at advanced stage, and colorectal cancer-specific mortality, overall and for whites and Blacks. Geographic clusters with higher-than-expected relative risk for each outcome of interest at the census tract level (HRCT) were identified and individual- and area-level descriptive statistics were calculated for Blacks, overall and by HRCT status. Adjusted hierarchical logistic regression analyses using backward stepwise elimination with model quasi-information criterion was performed to identify potential predictors of HRCTs for CRC incidence and advanced stage at diagnosis. Aim 2. Cross-sectional data from age-eligible adults, 50–75 years (N=357) participating in the ongoing CAP3 study was used to measure CRCS prevalence and adherence and region of birth (e.g., Caribbean-, African-, US-born). Prevalence and adherence were based on contemporaneous US Preventive Services Task Force guidelines. Descriptive statistics and adjusted prevalence and adherence proportions were calculated by region of birth. Adjusted logistic regression models were performed to assess the association between region of birth and overall CRCS and modality-specific adherence. Aim 3. To assess CRCS barriers, we conducted six sex-specific focus groups (n=3 female, n=3 male) with individuals, ages 45-75, who self-identified as Black (i.e., US-, Caribbean, or African-born) and were. Focus groups were held in person and via Zoom, recorded and transcribed verbatim. Codes were developed using coding consensus, co-occurrence, and comparison and open, axial, and selective coding rooted in grounded theory. Dedoose was used to determine CRCS barrier themes as well as general and modality-specific barriers by sex and by region of birth. Results Aim 1. In Philadelphia County, there were 4,641 CRC incident cases, of which 2,086 (44.9%) were non-Hispanic Black (NHB), and 2,555 (53.1%) were white. Mean age at diagnosis for CRC incidence (65.0 vs. 68.9 years), advanced stage at diagnosis (63.2 vs. 67.4 years), and colorectal cancer-specific mortality (67.5 vs. 71.7 years) was lower for Blacks compared to whites (p-value<0.001). Blacks were also diagnosed at a more advanced stage (25.0% vs. 22.4%, p-value=0.038) or unknown stage (8.01% vs. 5.64%, p-value=0.001). For each outcome, when compared to whites, higher proportions of Blacks lived in areas with higher proportions of markers of low socio-economic status and lower proportions of CRCS adherence. Geographic clusters at a higher-than-expected risk of CRC incidence were found in Northeast Philadelphia, North Philadelphia, West Philadelphia, and Southwest Philadelphia. Geographic clusters at a higher-than-expected risk of CRC diagnosed at an advanced stage and colorectal cancer-specific mortality overlapped and were in the North, Kensington, and Southwest neighborhoods of Philadelphia. Area-level NHB, the primary independent variable of interest, reduced the odds of HRCT for CRC incidence (OR: 0.971, 95% CI: 0.960, 0.983) and was not significantly associated with HRCT for advanced stage at diagnosis. In addition, after adjustment, for every one-unit increase in the percent of area-level foreign-born Blacks, there was 1.17-increased odds of being a HRCT for CRC incidence (95% CI: 1.07, 1.28). Similarly, there was a significant positive association with area-level foreign-born Black and being in a HRCT for advanced stage at diagnosis (OR: 1.15, 95% CI: 1.05, 1.26). Other area-level variables that were associated with HRCT for CRC incidence were median rent, percent of mortgaged housing units, and per capita income, which reduced the odds of being a HRCT; Percent of mortgaged housing units also reduced the odds of being a HRCT for advanced stage at diagnosis. Further, CRCS adherence reduced the odds of being a HRCT for advanced stage at diagnosis by approximately 15% (OR: 0.849, 95% CI: 0.791, 0.911). Aim 2. Respondents were 69.5% female, 43.3% married/living with a partner, and 38.4% had <$25,000 annual income. Overall, 78.2% reported past CRCS; however, stool test had the lowest prevalence overall (34.6%). Caribbean (95.0%) and African immigrants (90.2%) had a higher prevalence of overall CRCS compared to US-born Blacks (59.2%) (p-value <0.001). African immigrants were five times more likely to adhere to overall CRCS than US-born Blacks (OR: 5.25, 95% CI: 1.34, 20.6). Immigrants had higher odds of being adherent to colonoscopy (Caribbean=OR: 6.84, 95% CI: 1.49, 31.5; African =OR: 7.15, 95% CI: 1.27, 40.3) compared to US-born Blacks. Aim 3. The majority of focus group participants were 60–64 years old and 72% were immigrants (41% African-born, 31% Caribbean-born). Most participants had had CRCS, but 45% were non-adherent to national CRCS guidelines. Overall, lack of knowledge/awareness, fear, and a sense of feeling healthy and subsequently not seeing the need for CRCS emerged as overarching themes to CRCS barriers. General barriers differed by gender: for women lack of physician recommendation or explanation of CRCS was important and for men not knowing anyone with a history of CRCS was commonly cited. .” Differences in modality-specific barriers by gender were also noted. Barriers also differed by region of birth. US-born Blacks described lack of community advocacy promoting CRC and CRCS awareness as a barrier. African-born Blacks expressed lack of routine CRCS and utilization of preventive medicine in their native country as barriers. US- and Caribbean-born Black males, communicated that discussing CRCS was taboo, which was tied to hegemonic masculinity leading to a lack of conversations about CRC and CRCS. The use of traditional home remedies emerged as a barrier given respondents felt these remedies would aid in preventing CRC thereby reducing the need for CRCS. Immigrant Blacks also described limited insurance coverage due to their citizenship status. Conclusions This dissertation provides the first known insight into various outcomes across the colorectal cancer continuum for the heterogeneous Black population including the growing immigrant Black subgroups in Philadelphia County. To reduce CRC incidence and mortality, interventions and resources to increase CRCS uptake need to target geographic locations with higher percentages of foreign-born Blacks, lower CRCS adherence, and areas with worse socio-economic markers. Also, while Black immigrants had higher CRCS adherence compared to US-born Blacks, CRCS is still sub-optimal in the Black population. Further, CRCS barriers exist and differ by gender, and importantly, there are nuanced barriers by region of birth. Thus, efforts to increase CRCS should address the common and unique barriers and promote stool-based testing, as stool test adherence was low and focus group participants were unfamiliar with this modality as it is not widely promoted or available in clinical practice. In short, these findings across the colorectal cancer continuum should be taken into account for resource allocation and when designing targeted or tailored interventions to promote CRCS uptake for the heterogeneous Black population, which would reduce CRC incidence, late-stage diagnosis, and mortality. / Epidemiology

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Crohn's disease (CD) and colorectal cancer (CRC) are major disorders of the intestine. Inflammation in CD often precedes fibrosis and stricture formation, and is linked to increased cancer risk. Hypoxia is a common feature of inflammation and CRC that can severely compromise the effectiveness of current therapy regimes including chemo-radiotherapy and maintenance of remission in CD patients. MicroRNAs (miRNAs) are key regulatory molecules involved in cellular proliferation, apoptosis and fibrosis, which are all modulated by hypoxia. This thesis aims to understand the role of miRNAs in these two intestinal diseases. Microarray profiling identified differentially expressed miRNA in the intestinal mucosa overlying strictured and non-strictured CD tissue samples and in six CRC cell lines cultured in hypoxic conditions compared to normoxia. Validation experiments using qRT-PCR confirmed the differential expression of miR-29a, -29b, -29c, -34a, -493* and -708 in CD mucosa and miR-21, -210, -30d, -320a, -320b and -320c in CRC cell lines. Functionally, over-expression of miR-29b in CD intestinal fibroblasts modulated the down-regulation of collagen I and III transcripts and collagen III protein in a TGF-β-dependent manner. Furthermore, miR-29b induced indirectly the expression of the anti-apoptotic protein Mcl-1 via the cytokines IL-6 and IL-8. A positive correlation between miR-210 and the hypoxia marker CAIX was found in CRC tissue in vivo. Furthermore, HCT116 cells cultured under hypoxia were more resistant to the chemotherapy drug 5-FU than cells grown under normoxia. Knockdown of miR-21 or miR-30d under hypoxia may sensitise CRC cells to 5-FU. CRC cell lines grown under hypoxic conditions present an altered cellular metabolic profile compared to their normoxic counterparts. This thesis has showed that critical miRNAs have a functional role in the progression of two important diseases of the intestine. The work presented highlights the potential of miRNAs as biomarkers and therapeutic targets to improve the clinical management of patients with digestive diseases.

Background: The five year overall survival rate for colorectal cancer (CRC) patients varies between 38.8% and 59.9%. Selecting patients who are likely to respond to therapy remains a clinical and pathological challenge, hence the need for predictive and prognostic biomarkers. The objectives of this study were: 1) to establish which genes were differentially expressed with respect to sensitivity to treatment, 2) to integrate the list of differentially expressed genes with copy number to systematically identify predictive biomarkers, and 3) to establish which genes are commonly gained in the panel of CRC cell lines. As proof of concept of the approach the copy number variations of the identified genes were assessed in a cohort of Dukes’ A and B cancers, in order to analyse the likelihood of these genes acting as useful biomarkers. Methods: Cell viability assays were carried out on a panel 15 CRC cell lines. IC50s were measured for 5-fluoruracil (5-FU), oxaliplatin (L-OHP), and BEZ-235, a PI3K/mTOR inhibitor. We carried out a systematic array-based survey of gene expression and copy number variation in CRC cell lines, and compared these to responses to different treatments. Cell lines were profiled using array comparative genomic hybridisation (aCGH; NimbleGen 135k), Illumina gene expression analysis, reverse phase protein arrays (RPPA), and targeted sequencing of KRAS hotspot mutations. The associations between the biological variables and drug sensitivity were assessed using correlation coefficients, chi-square analysis, and the Mann Whitney-U test. Tissue microarrays (TMA) were constructed for a cohort of CRC patients (n=118) and TRIB1 and MYC amplifications were measured using fluorescence in situ hybridisation (FISH). The protein expression for trib1 and 14 associated biomarkers were investigated using Automated Quantitative Analysis (AQUA) and analysed using the Pearson’s correlation coefficient. Results: Twenty-three regions were frequently gained, and fourteen regions were lost across the cell line panel. Gains were observed at 2p, 3q, 5p, 7p, 7q, 8q, 12p, 13q, 14q, and 17q, and losses at 2q, 3p, 5q, 8p, 9p, 9q, 14q, 18q, and 20p. Frequently gained regions contained EGFR, PIK3CA, MYC, SMO, TRIB1, FZD1, and BRCA2, while frequently lost regions contained FHIT and MACROD2. Gene enrichment analysis showed that differentially expressed genes with respect to treatment response were involved in Wnt signalling, EGF receptor signalling, apoptosis, cell cycle, and angiogenesis. Stepwise integration of copy number and gene expression data yielded 47 candidate genes that were significantly correlated (corrected p-value ≤0.05). Differentially expressed genes common to all three treatment responses included AEBP2, DDX56, MRPL32, MRPS17, MYC, NSMCE2, and TBRG4. TRIB1 (n=76) and MYC (n=81) were amplified (FISH score ≥1.8) in 14.5% and 7.4% of the CRC cohort, respectively. TRIB1 and MYC amplifications were significantly correlated (corrected p-value ≤ 0.0001). Trib1 protein expression in the patient cohort was significantly correlated (corrected p-value ≤ 0.01) with protein expression of pErk, Akt, and Caspase 3. Conclusions: The CRC in-vitro model was used effectively in this study for discovery of both predictive and prognostic biomarkers. A set of candidate predictive biomarkers for 5-FU, L-OHP, and BEZ235 have been described, worthy of further study. Amplification of the putative oncogene TRIB1 has been assessed for the first time in a cohort of CRC patients. Inhibition of TRIB1 may be a synthetic lethal approach when MYC amplifications are present, which requires further clinical and experimental validation.

Colorectal cancer (CRC) is common, with >1 million annual incidence worldwide and is associated with significant morbidity and mortality. Prevention is a particularly appealing strategy to combat CRC, but there is a paucity of well-founded mechanistic research in the area. CRC is a complex disorder, with both genetic and environmental factors influencing incidence. The heritable component of CRC variance is estimated to be ~35%, with ~5% due to highly penetrant mutations. Common genetic variance likely makes up the majority of the heritable component, yet a large proportion of heritability remains unexplained. One possible explanation is gene-environment interactions (GxE) where-by both genetic and environmental factors interact to influence risk. Observational data implicate vitamin D as an environmental risk factor in CRC aetiology and in-house data indicates that genotype at the GWAS identified CRC risk locus rs9929218 influences this association, i.e. a GxE. The rs9929218 locus is intronic within CDH1, a tumour suppressor gene, and present evidence suggests a gene-environment interaction model of vitamin D-induced CDH1 transcription dependent on genotype at the rs9929218 locus and mediated by VDR and FOXO transcription factors and SIRT1, a FOXO regulator. To test this model, two broad approaches were employed - an observational approach to assess the association between human plasma vitamin D status, rs9929218 genotype and normal colorectal mucosa CDH1 expression and an interventional approach treating cell lines, organoids and human subjects with vitamin D to assess genotype-specific effects. Observational analysis of vitamin D level (25OHD) in CRC cases identified a significant influence of age, gender, BMI and selected vitamin D pathway genetic variation, while analysis of 424 normal colorectal mucosa samples from CRC cases and cancer-free subjects demonstrated strong sampling, gender, age and site differences in gene expression. 25OHD was not significantly associated with mucosa gene expression at individual gene level. However, association with a number of pathways relevant to tumourigenesis, including 'cell adhesion', 'migration' and 'cell death' was seen, providing possible mechanism to the published observational data. Circulating 25OHD level was not associated with mucosa CDH1 expression, yet crucially, analysis demonstrated a strong additive gene-environment interaction effect between plasma 25OHD, the rs9929218 genotype and NM expression of VDR, FOXO and SIRT1 explaining ~70% of the variance of mucosal CDH1 expression. The interventional approach first investigated vitamin D effects on CRC cell lines and human colorectal mucosa organoids. Calcitriol, the active form of vitamin D, induced CDH1 expression in 4 CRC cell lines, with interaction effects explaining 66% of the variance of CDH1 expression. CDH1 induction was replicated in human colorectal epithelial organoids, a non-aberrant tissue model, while gene enrichment analysis from both cells and organoids implicate vitamin D in a number of processes relevant to CRC tumourigenesis including regulation of cell proliferation, differentiation, migration and apoptosis, consistent with the pleiotropic effects of vitamin D reported in the published literature. Finally, a novel human intervention study was undertaken to investigate the impact of vitamin D supplementation in human blood and rectal mucosa. Short-term high-dose supplementation of 50 participants significantly induced CDH1 expression in rectal mucosa, with significant gene interaction effects between 25OHD, rs992818 genotype and CDH1, VDR and FOXO expression, thus independently replicating the same gene interaction effects seen in the human observational study. Meanwhile, transcriptome profiling identified numerous pathways relevant to tumourigenesis significantly enriched after supplementation, validating several pathways also associated with vitamin D status in the observational study. In summary, the research presented in this thesis demonstrates that vitamin D treatment of cells, epithelial organoids and human subjects induces CDH1 expression, and that strong gene interaction effects involving the colorectal cancer risk locus rs9929218 modulate this effect. FOXO transcription factors influence the gene interaction effect, consistent with the proposed model of ligand dependent regulation of FOXO by VDR and transcription activation of the CDH1 gene by the FOXO complex dependent on rs9929218 genotype. These data provide support for rectal CDH1 expression as an intermediate biomarker for vitamin D chemopreventive studies and suggest that gene environment interactions underlie some of the missing heritability of CRC.

Introduction: This study describes the patterns of use and processes of care following colonic stent insertion for patients with colorectal cancer (CRC) in clinical practice. Methods: Ontario residents who had a colonic stent placed for CRC between 2000–2009 were identified using linked administrative databases. Baseline patient, physician, and institutional characteristics were extracted. The cohort was followed for death and health services utilization post-stent. Results: Two hundred twenty-five patients were identified. Median overall survival post-stent insertion was 199 days (interquartile range [IQR] 153-282). Eighty-five (38%) patients required a subsequent intervention (abdominal surgery, restenting, and/or dilatation). Median intervention-free survival was 75 days (IQR 59-91). Following stent insertion, the average rate of ER visits was 2.4 visits per person-year of follow up (95% CI, 2.2-2.7) and the overall average days spent in hospital was 19 inpatient days per person-year (95% CI, 18-19). Conclusions: In clinical practice, many patients required another intervention shortly after stent insertion; however, the rate of post-stent ER visits and inpatient hospital days was low.

Introduction: This study describes the patterns of use and processes of care following colonic stent insertion for patients with colorectal cancer (CRC) in clinical practice. Methods: Ontario residents who had a colonic stent placed for CRC between 2000–2009 were identified using linked administrative databases. Baseline patient, physician, and institutional characteristics were extracted. The cohort was followed for death and health services utilization post-stent. Results: Two hundred twenty-five patients were identified. Median overall survival post-stent insertion was 199 days (interquartile range [IQR] 153-282). Eighty-five (38%) patients required a subsequent intervention (abdominal surgery, restenting, and/or dilatation). Median intervention-free survival was 75 days (IQR 59-91). Following stent insertion, the average rate of ER visits was 2.4 visits per person-year of follow up (95% CI, 2.2-2.7) and the overall average days spent in hospital was 19 inpatient days per person-year (95% CI, 18-19). Conclusions: In clinical practice, many patients required another intervention shortly after stent insertion; however, the rate of post-stent ER visits and inpatient hospital days was low.

Elevated deoxycholic acid (DCA) and mutations in the Adenomatous Polyposis Coli (APC) tumor suppressor gene have been associated with increased risk of colorectal cancer (CRC). Chronic inflammation has also been associated with increased risk of CRC. It is unclear if DCA mediates inflammation in the normal or transformed colonic mucosa. The status of APC was manipulated in human CRC cell lines to study the role of DCA mediated inflammation. The chemotactic cytokine, CXCL8, was used as a marker of inflammation. Addition of DCA to the HT29-parental cell line with mutant-APC increased the steady state mRNA and protein levels of CXCL8. Conversely, addition of DCA to the HT29-APC cell line with wild type-APC was protective for increased steady state RNA and protein levels of CXCL8. DCA activated transcription factors which had binding regions in the CXCL8 5’-promoter. To elucidate the mechanism of induction, the 5’-promoter of CXCL8 was investigated. DCA increased promoter-reporter activity of the CXCL8 gene in HT29-parental cell line but wild type-APC blocked this effect. Chromatin immunoprecipitation (ChIP) revealed that DCA activated transcription factors, AP-1 and NF-κB were bound to the 5’-promoter of CXCL8. The transcription factor, β-catenin, was also bound to the 5’-promoter of CXCL8. Phenotypic effects were measured. Increased CXCL8 lead to matrix metalloproteinase-2 (MMP-2) production and increased invasion by HT29-parental cells on laminin coated filters. The DCA-mediated invasion was blocked by antibody directed against CXCL8 and wild type- APC. Therefore DCA-mediated inflammation occurs in transformed colonic epithelium and increases the invasive phenotype of CRC cells by CXCL8.