Glioblastoma is the commonest form of brain neoplasm and among the most malignant forms of cancer. The identification of a subpopulation of self-renewing and multipotent cancer stem cells within glioblastoma has revealed a novel cellular target for the treatment of this disease. The role of developmental cell signaling pathways in these cell populations remains poorly understood. Herein, we examine the role of the Notch signaling pathway in glioblastoma stem cells. In this thesis we have demonstrated that the canonical Notch pathway is active in glioblastoma stem cells and functions to inhibit neuronal lineage commitment in a subset of patient derived glioblastoma stem cells in vitro. Gamma secretase (γ-secretase) small molecule inhibitors or dominant-negative co-activators inhibit glioblastoma stem cell proliferation and induce neuronal lineage commitment in a fashion that synergizes with Wingless pathway activation via GSK-3β blockade. Our data suggest that subsets of patient samples show a Notch gene expression profile that predicts their abilities to undergo neuronal lineage differentiation in response to γ-secretase small molecule inhibitors. Additionally, the data suggests that Notch may perturb the relative fractions of cells undergoing symmetric division, in favour of asymmetric division, limiting clonal expansion from single cells. These data may have important implications for treating human glioblastoma, and suggest that in addition to inhibition of proliferation, influencing lineage choice of the tumor stem cells may be a mechanism by which these tumors may be pharmacologically inhibited.

DKK1, SFRP1, WIF-1, and Wnt5a encode Wnt pathway genes that are frequently silenced by promoter hypermethylation in colorectal cancer. Despite attractive biological consequences of these events, it is unclear whether they contribute to patient prognostication or may influence tumour cell biology within distinct patient subsets. I sought to determine the prognostic roles of these methylation events in a large cohort of colorectal carcinomas from Ontario and Newfoundland. Methylation was quantified and associated with patient clinicopathlogical features. Methylation was present in cancer tissue. DKK1, Wnt5a, and SFRP1 were strongly and independently associated with tumour subtype in a manner that suggested subtype-specific activity of Wnt signaling. Methylation of DKK1 was a borderline prognosticator of favourable outcome. These results offer intriguing insight into subtype-specific biology and lead to a proposed model whereby methylation-induced Wnt bias may contribute to patient outcome.

The evolution of prostate cancer from normal epithelium via the preneoplastic lesion of high-grade prostatic intraepithelial neoplasia to invasive carcinoma is characterised by a number of particular genomic abnormalities that are predominantly generated in the preneoplastic phase. Whilst there are numerous candidates for the cause of these alterations, telomere dysfunction is thought to be a major contributor. Telomeres are the terminal ends of human chromosomes, and when dysfunctional can lead to break-fusion-bridge cycles and multi-polar mitoses that generate numerical and structural chromosomal instability. The results presented reinforce the association of telomere dysfunction with the generation of certain markers of genomic instability such as abnormalities of the arms of chromosome 8. Furthermore, this work clarifies that the TMPRSSS2-ERG aberrations are not telomere related phenomena and are associated with a genomic deletion in a proportion of cases. Similarly, the PTEN microdeletions did not appear to have an association with telomere attrition. A previously unrecognised association between the telomere length in various types of prostatic epithelia and adjacent stroma is defined, suggesting evidence of a micro-environmental field effect in the generation of prostatic neoplasia. Finally, when examined retrospectively, it appears that telomere attrition, both in the HPIN epithelium and the stroma has independent prognostic value in the diagnosis of prostate cancer after a previous diagnosis of HPIN. Taken together, the research presented suggests important avenues for further research to determine the nature of barriers to the evolution of prostatic carcinogenesis such as oncogene- and telomere-induced senescence that may be exploited for therapeutic gain. These understandings may also help tailor management for prostate cancer such as risk stratification for men with HPIN and the use of targeted agents such as AKT inhibitors and telomerase inhibitors. In more advanced disease, translational application of this work has enabled a clinical trial of cytarabine in the treatment of metastatic hormone refractory prostate cancer.

Introduction: This study evaluated long-term outcomes of young survivors of colorectal cancer including survival, development of acute illnesses, and childbirth. Methods: Persons aged 20-44 diagnosed with colorectal cancer and surviving at least five years were identified using the Ontario Cancer Registry and compared to randomly selected controls. Outcomes included death, admission to hospital for acute illness, and childbirth in women, determined by linkage to provincial administrative data. Results: There were 917 cancer survivors in this study. Survivors were more likely to die (HR 8.2, 95%CI [5.8, 11.6]), and require admission to hospital for acute illness (rate ratio 3.4, 95%CI [2.9, 4.1]) than controls. There was no difference in admissions for childbirth in women (HR 0.6, 95%CI [0.3, 1.4]). Conclusions: Five-year survivors of colorectal cancer remain at high risk of long-term death and illness. Aggressive surveillance for recurrent malignant disease is necessary to mitigate risk of death.

The evolution of prostate cancer from normal epithelium via the preneoplastic lesion of high-grade prostatic intraepithelial neoplasia to invasive carcinoma is characterised by a number of particular genomic abnormalities that are predominantly generated in the preneoplastic phase. Whilst there are numerous candidates for the cause of these alterations, telomere dysfunction is thought to be a major contributor. Telomeres are the terminal ends of human chromosomes, and when dysfunctional can lead to break-fusion-bridge cycles and multi-polar mitoses that generate numerical and structural chromosomal instability. The results presented reinforce the association of telomere dysfunction with the generation of certain markers of genomic instability such as abnormalities of the arms of chromosome 8. Furthermore, this work clarifies that the TMPRSSS2-ERG aberrations are not telomere related phenomena and are associated with a genomic deletion in a proportion of cases. Similarly, the PTEN microdeletions did not appear to have an association with telomere attrition. A previously unrecognised association between the telomere length in various types of prostatic epithelia and adjacent stroma is defined, suggesting evidence of a micro-environmental field effect in the generation of prostatic neoplasia. Finally, when examined retrospectively, it appears that telomere attrition, both in the HPIN epithelium and the stroma has independent prognostic value in the diagnosis of prostate cancer after a previous diagnosis of HPIN. Taken together, the research presented suggests important avenues for further research to determine the nature of barriers to the evolution of prostatic carcinogenesis such as oncogene- and telomere-induced senescence that may be exploited for therapeutic gain. These understandings may also help tailor management for prostate cancer such as risk stratification for men with HPIN and the use of targeted agents such as AKT inhibitors and telomerase inhibitors. In more advanced disease, translational application of this work has enabled a clinical trial of cytarabine in the treatment of metastatic hormone refractory prostate cancer.

Introduction: This study evaluated long-term outcomes of young survivors of colorectal cancer including survival, development of acute illnesses, and childbirth. Methods: Persons aged 20-44 diagnosed with colorectal cancer and surviving at least five years were identified using the Ontario Cancer Registry and compared to randomly selected controls. Outcomes included death, admission to hospital for acute illness, and childbirth in women, determined by linkage to provincial administrative data. Results: There were 917 cancer survivors in this study. Survivors were more likely to die (HR 8.2, 95%CI [5.8, 11.6]), and require admission to hospital for acute illness (rate ratio 3.4, 95%CI [2.9, 4.1]) than controls. There was no difference in admissions for childbirth in women (HR 0.6, 95%CI [0.3, 1.4]). Conclusions: Five-year survivors of colorectal cancer remain at high risk of long-term death and illness. Aggressive surveillance for recurrent malignant disease is necessary to mitigate risk of death.

One of the best ways to diagnose breast cancer early or to predict therapeutic response is to use serum biomarkers. Unfortunately, for breast cancer, we do not have effective serological biomarkers. We hypothesized that novel candidate tumor markers for breast cancer may be secreted or shed proteins that can be detected in tissue culture supernatants of human breast cancer cell lines. A two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) strategy was utilized to identify and compare levels of extracellular and membrane-bound proteins in the conditioned media. Proteomic analysis of the media identified in excess of 600, 500 and 700 proteins in MCF-10A, BT474 and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6 and 10 (ranging in concentration from 2-50 µg/L), as validated by ELISA and confidently identified HER-2/neu in BT474 cells. Sub-cellular localization was determined based on Genome Ontology (GO) for the 1,139 proteins, of which 34% were classified as extracellular and membrane-bound. Tissue specificity, functional classifications and label-free quantification were performed. The levels of eleven promising molecules were measured in biological samples to determine its discriminatory ability for control versus cases. This screen yielded activated leukocyte cell adhesion molecule (ALCAM) as a promising candidate. The levels of ALCAM, in addition to the classical breast cancer tumor markers carbohydrate antigen 15-3 (CA 15-3) and carcinoembryonic antigen (CEA) were examined in 300 serum samples by quantitative ELISA. All three biomarkers effectively separated cancer from non-cancer groups. ALCAM, with area under the curve (AUC) of 0.78 [95% CI: 0.73, 0.84] outperformed CA15-3 (AUC= 0.70 [95% CI: 0.64, 0.76]) and CEA (AUC= 0.63 [95% CI: 0.56, 0.70]). The incremental values of AUC for ALCAM over that for CA15-3 were statistically significant (Delong test, p <0.05). Serum ALCAM appears to be a new biomarker for breast cancer and may have value for disease diagnosis.

Paediatric ependymomas are the third most common childhood brain cancer and represent a prognostic and therapeutic challenge. Previous evidence suggests that telomerase, a ribonucleoprotein critical in permitting limitless growth potential, may serve as both a prognostic marker and therapeutic target. Immunohistochemical analysis (n=198) and enzymatic detection (n=25) of telomerase was performed to determine prevalence and prognostic potential. The telomerase inhibitor Imetelstat was used to study telomerase inhibition in paediatric ependymoma cell lines, tumour initiating cells (TICs) and both subcutaneous and intracranial xenografts. Telomerase activity was detected in 76% of primary ependymomas and was associated with a reduced five-year progression-free survival (30% vs 75%). Telomerase inhibition in vitro resulted in shortened telomeres, increased senescence, growth inhibition and reduced self-renewal capacity. In vivo, Imetelstat shortened telomeres and reduced subcutaneous tumour volume by 40% compared to control mice. Therefore, telomerase may serve as an ideal prognostic marker and therapeutic target in paediatric ependymoma.

Low Dose Metronomic (LDM) chemotherapy, combined with VEGF pathway inhibitors, is a highly effective strategy to coordinately inhibit angiogenesis and tumor growth. We have tested the efficacies of daily oral LDM topotecan alone and in combination with pazopanib, in three pediatric extracranial solid tumors mouse models. We also investigated the effect of prolonged combination therapy with the combination on tumor behavior in a neuroblastoma mouse xenograft model. In-vitro dose-response study of topotecan and pazopanib was conducted on several cell lines. In-vivo antitumor efficacies of drugs, as single agents and combination, were tested in immunodeficient mice models. For studying the mechanisms of resistance to our therapy, a time-response study (28, 56 and 80 days) was conducted in SK-N-BE(2) xenografts model, treated in same way as earlier. In vitro, topotecan caused a dose-dependent decrease in viabilities of all cell lines, while pazopanib did not. In vivo, the combination of topotecan and pazopanib demonstrated significant anti-tumor activity compared to the respective single agents in all models. Reductions in the levels of viable Circulating Endothelial Progenitors and/or Circulating Endothelial Cells and tumor microvessel density were correlated with tumor response and therefore confirmed the antiangiogenic activity of the regimens. However, the combination also caused significantly higher myelotoxicity than single agents. Pharmacokinetic study did not reveal any interaction between the two co-administered drugs. In the time-response study, we found that only combination treated animals survived till 80 days. However, tumors in these animals started growing gradually after 50 days. Unlike single agents, all three durations of combination treatment significantly lowered tumor microvessel densities, compared to the control. However, tumors treated with the combination for 56 and 80 days had higher pericyte coverage. The combination increased the hypoxia, angiogenic expression and proliferative index and caused metabolic reprogramming of tumor cells. We conclude that the combination of LDM topotecan and pazopanib has superior efficacy than either single agents, which is attributed to superior antiangiogenic activity. However, prolonged treatment with the combination can have additive myelotoxicity and may encounter adaptive resistance associated with metabolic reprogramming and increased proliferation of the tumor cells.

One of the best ways to diagnose breast cancer early or to predict therapeutic response is to use serum biomarkers. Unfortunately, for breast cancer, we do not have effective serological biomarkers. We hypothesized that novel candidate tumor markers for breast cancer may be secreted or shed proteins that can be detected in tissue culture supernatants of human breast cancer cell lines. A two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) strategy was utilized to identify and compare levels of extracellular and membrane-bound proteins in the conditioned media. Proteomic analysis of the media identified in excess of 600, 500 and 700 proteins in MCF-10A, BT474 and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6 and 10 (ranging in concentration from 2-50 µg/L), as validated by ELISA and confidently identified HER-2/neu in BT474 cells. Sub-cellular localization was determined based on Genome Ontology (GO) for the 1,139 proteins, of which 34% were classified as extracellular and membrane-bound. Tissue specificity, functional classifications and label-free quantification were performed. The levels of eleven promising molecules were measured in biological samples to determine its discriminatory ability for control versus cases. This screen yielded activated leukocyte cell adhesion molecule (ALCAM) as a promising candidate. The levels of ALCAM, in addition to the classical breast cancer tumor markers carbohydrate antigen 15-3 (CA 15-3) and carcinoembryonic antigen (CEA) were examined in 300 serum samples by quantitative ELISA. All three biomarkers effectively separated cancer from non-cancer groups. ALCAM, with area under the curve (AUC) of 0.78 [95% CI: 0.73, 0.84] outperformed CA15-3 (AUC= 0.70 [95% CI: 0.64, 0.76]) and CEA (AUC= 0.63 [95% CI: 0.56, 0.70]). The incremental values of AUC for ALCAM over that for CA15-3 were statistically significant (Delong test, p <0.05). Serum ALCAM appears to be a new biomarker for breast cancer and may have value for disease diagnosis.