Pancreatic adenocarcinoma (PDAC) putatively arises from the pancreatic duct, thus usage of the normal human pancreatic duct epithelial (HPDE) cell line is an ideal model to examine the successive accumulation of genetic alterations involved in carcinogenesis. KRAS mutations have been reported in 90% of PDACs. Oncogenic KRAS elicits activation of downstream pathways involved in survival, motility, and cell cycle progression. KRASG12V introduction in the HPDE cell line upregulates Lipocalin-2 (LCN2) expression. LCN2 has been identified in numerous carcinomas and is associated with survival, tumorigenicity, and invasion. In this work, LCN2 was found to be commonly expressed in high grade pancreatic duct neoplastic precursor lesions and PDAC illustrating its potential as a biomarker. Moreover, in vitro and in vivo studies demonstrate that high LCN2 expression promotes gemcitabine resistance, MMP-9 activity, angiogenesis, and tumorigenicity. Loss of Smad4 function is found in 55% of PDAC cases. Smad4 is a critical component in the TGF-β signaling which mediates the transcription of genes involved in processes such as cell cycle arrest, apoptosis, and invasion. This work examined the consequences of KRASG12V expression and Smad4 loss in the HPDE model. Cellular invasion was promoted by KRASG12V expression or knocking down Smad4 by 80% in the HPDE model. A TGF-β resistant HPDE cell line, TβR, was shown to lack Smad4 expression due to deletion, promoter methylation, and nonsense mutation. KRASG12V expression in the TβR model (TβR KRAS) promoted neoplastic transformation and tumour formation in immunodeficient mice with complete penetrance. Smad4 expression in the TβR KRAS cell line reinstated TGF-β signaling, delayed tumour formation, and decreased metastatic spread. This study provides evidence that Smad4 acts as a restriction point in the transformation of HPDE cells. Overall, this work examines the contribution of genes involved in transformation, and identifies a potential therapeutic and diagnostic biomarker in PDAC.

Currently, the intensive chemotherapy remains the first line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although these regimens have significantly improved patient outcomes, their use is associated with debilitating morbidities and fatal relapses, highlighting the great need in new agents that target essential survival signals in leukemia. Thus, the overall goal of my project was to gain insights into the signaling abnormalities that regulate aberrant proliferation and survival of B-ALL cells in an effort to identify novel targets in this malignancy. This study demonstrated that pre-B cell receptor (pre-BCR)-independent spleen tyrosine kinase (SYK) activity was required for the survival and proliferation of a p53-/-PrkdcSCID/SCID mouse model of B-ALL. I extended this discovery to human disease, demonstrating that SYK was activated in primary B-ALL, independent of the pre-BCR expression. The small molecule SYK inhibitor fostamatinib (fosta) significantly attenuated proliferation of 79 primary diagnostic B-ALL samples at clinically achievable concentrations. Importantly, fosta treatment reduced dissemination of engrafting B-ALL cells into the spleen, liver, kidney and central nervous system (CNS) in a NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ xenotransplant model of B-ALL. Analysis of signaling abnormalities using a high-throughput phospho-flow cytometry platform demonstrated that pediatric and adult B-ALL samples exhibit variable basal activation of BCR, iii PI3K/AKT/mTOR, MAPK and JAK/STAT pathways. Importantly, we identified that fosta-mediated inhibition of SYK, PLC2, CRKL and EIF4E phosphorylation in B-ALL was predictive of its anti-leukemic activity, and was distinct from the cellular actions of other small molecule inhibitors of key nodal signaling pathways. Examination of molecular mechanism of fosta action by gene expression profiling revealed transcriptional effects of fosta treatment that included, most notably, potent inhibition of pathways involved in lymphocyte activation and inflammation. In conclusion, this study demonstrates that SYK signaling is crucial for B-ALL survival and provides detailed characterization of cellular and molecular mechanisms of fosta action in B-ALL. These data argue in favor of testing small molecule SYK inhibitors in pediatric and adult B-ALL.

Ovarian high-grade serous cancer (HGSC) is the most lethal gynecologic malignancy and well-characterized models may improve patient outcomes. Patient-derived xenografts (PDXs) recapitulate disease heterogeneity; however, to be useful in predicting response to novel chemotherapeutics, they must reflect the response of the donor tissue to standard chemotherapy. The objectives of this study were: first, to evaluate the response of PDXs’ to platinum therapy and compare this response to that of the donor; and second, to determine whether treatment with chemotherapy enriches for tumourigenic cells. Eighteen samples formed tumours in the mammary fat pads of NOD-Scid-IL2Rγnull mice and were treated with Carboplatin. There was a 100% concordance between sample status and PDXs response to chemotherapy. HGS histology was confirmed for all cases. A conclusion regarding post-chemotherapy tumourigenicity could not be made due to inadequate statistical power. PDXs represent useful tools for evaluation of novel therapies and identification of patients who are platinum-resistant/sensitive.

Background: This thesis is composed of three studies. In the first paper, we tested the association of metformin use with prostate cancer incidence. In the second paper, we examined the association of metformin use with all-cause and prostate cancer specific mortality. The final paper explored the benefit of detailed pathology review to predict mortality among diabetic men with prostate cancer. Methods: A total of 5306 incident diabetic men older than 66 who subsequently developed prostate cancer were identified using the Ontario Diabetes Database and the Ontario Cancer Registry between 1994-2008. The association of metformin use and risk of prostate cancer and its grade was tested with a nested case-control design using a conditional logistic regression model. We used a cohort design with a time dependent Cox-proportional hazard model to examine the association of metformin use and mortality. Finally, we employed a c-statistic and Net Reclassification Improvement analysis to study the impact of pathology abstraction on predicting mortality. Results: The data suggest metformin use was not associated with the risk of prostate cancer or its grade at presentation. However, each additional 6 month of metformin use was associated with a 24% decrease in prostate-cancer-specific and 8% decrease in all-cause mortality. Pathology abstraction improved the accuracy in predicting all-cause and prostate-cancer specific mortality. Conclusions: In our study metformin use was not associated with a decreased risk of prostate cancer, but had a significant impact on all-cause and prostate cancer specific mortality. These results may serve as proof of concept in designing an interventional study of metformin to delay progression in prostate cancer.

High-grade serous ovarian cancer (HG-SOC) is the most lethal gynecologic malignancy. Although most respond to initial therapy, the vast majority of patients eventually recur and die of their disease. Understanding intra-tumor cellular heterogeneity and inter-patient variability is necessary to effectively cure HG-SOC. The work described in this thesis should help to speed the progress of ovarian cancer research in several ways. First, I generated a robust xenograft model that recapitulates the cellular heterogeneity of HG-SOC. In addition, I performed gene expression profiling on a subset of xenografts and showed that they recapitulate the inter-patient diversity of this disease. Second, I applied this model to pre-clinical testing of a folate-targeted imaging agent and showed that it can identify metastatic studding by PET/CT and fluorescence imaging. Using my xenograft model, I investigated the properties of tumor-initiating cells (TIC) and demonstrated that TIC in HG-SOC are rare. Furthermore, although CD133 marks most TIC, heterogeneity in the phenotype is observed within individual tumors and between different patients. Finally, I used a transformative technology, CyTOF, to develop a novel pipeline for prioritization of candidate TIC markers, as well as for characterization of cellular heterogeneity in primary HG-SOC samples.

The oncogenic transcription factor STAT3 is aberrantly activated in over 70% of human tumours, including Multiple myeloma (MM). The present studies use both genetic and chemical tools to validate STAT3 as a therapeutic target, and demonstrate the anti-MM activity of a novel small molecule STAT3 inhibitor, BP-4-018. We show that shRNA-mediated STAT3 knockdown induces apoptosis in human myeloma cell lines (HMCLs). We translate these findings to a therapeutically relevant setting by demonstrating the broad anti-MM activity of BP-4-018 against HCMLs and primary patient samples, and demonstrate that BP-4-018 remains active against HMCLs co-cultured with bone marrow stroma. Inhibiting STAT3 via shRNA knockdown and BP-4-018 suppresses STAT3 transcriptional activity and down-regulates anti-apoptotic and proliferative STAT3 target genes. Finally, we show that BP-4-018 has activity in vivo, both alone and combined with subtherapeutic doses of bortezomib, without significant toxicities. Taken together, these data support the utility of STAT3 inhibitors for MM treatment.

According to the cancer stem cell hypothesis a subpopulation of cells within a tumour has the capacity to sustain its growth. These cells are termed cancer stem cells, and are most simply defined as the cells within a primary tumour that can self-renew, differentiate and regenerate a phenocopy of that cancer when transplanted in vivo. Cancer stem cells have now been prospectively identified from numerous human tumours and are actively sought in many cancer types, both clinical and experimental. The cancer stem cell hypothesis remains controversial, with evidence both supporting and challenging its existence in human tumours and in animal models of disease. Here we prospectively identify and study brain cancer stem cells in clinically representative mouse models of the medulloblastoma and glioma. Cancer stem cells from both mouse brain tumour types are prospectively enriched by fluorescent activated cell sorting freshly dissociated cells for the surface antigen CD15, display a neural precursor phenotype, exhibit the hallmark stem cell characteristics of self-renewal and multilineage differentiation, and regenerate a phenocopy of the original tumour after orthotopic transplantation. Additionally, novel mouse medulloblastoma and glioma cancer stem cell lines were established and studied in vitro as adherent cultures in the same serum-free media conditions that support the growth of normal neural stem cells. When mouse and human glioma stem cell lines were compared, many novel molecular mediators of the tumour phenotype were identified, as were chemical compounds that selectively inhibit their growth. Our results have important implications regarding the cancer stem cell hypothesis, the mechanisms that drive brain tumour stem cell growth and the therapeutic strategies that may prove effective for the treatment of glioma and medulloblastoma.

The orphan nuclear receptor EAR-2 (NR2F6) is a gene that I previously found to be expressed at a higher level in clonogenic leukemia single cells than in leukemia cells that can not divide. For this thesis I undertook to perform the first investigations of the roles EAR-2 may play in normal haematopoiesis and in the pathogenesis of acute myelogenous leukaemia. Here, I show that EAR-2 is overexpressed in the bone marrow of patients with MDS, AML and CMML compared to healthy controls and that EAR-2 is a gatekeeper to hematopoietic differentiation. Over-expression of EAR-2 prevents the differentiation of cell lines, while knock down induces their spontaneous differentiation. In vitro, primary bone marrow cells that over-express EAR-2 do not differentiate into granulocytes in suspension culture, but have greatly extended replating capacity in colony assays. In vivo, overexpression of EAR-2 in a chimeric mouse model leads to a condition that resembles myelodysplastic syndrome characterised by hypercellular bone marrow, an increase in blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice. Furthermore, animals that are transplanted with grafts of sorted bone marrow develop a rapidly fatal leukemia that is characterized by pancytopenia, enlargement of the spleen, infiltration of blasts into the spleen, liver and peripheral blood. Interestingly, development of leukemia is preceded by expansion of the stem cell compartment. Overexpression of EAR-2 increases the maintenance of KSL primitive bone marrow cells in ex vivo suspension culture, while knockdown of EAR-2 induces rapid differentiation of KSL cells into granulocytes. These data establish that EAR-2 is a novel oncogene that regulates hematopoietic cell differentiation. Furthermore, I show that EAR-2 is also a novel negative regulator of T-cell lymphopoiesis, and demonstrate that down-regulation of EAR-2 is important for the survival, proliferation and differentiation of T-cell progenitors. Overall, this work establishes that expression of EAR-2 is an important determinant of cell fate decisions in the hematopoietic system.

Aberrant DNA methylation in gene promoters causes gene silencing and is a common event in prostate cancer development and progression. While commonly identified methylated genes have been analyzed for their potential clinical utility in a variety of cancers, few studies have attempted a genome-wide methylation approach to discover new and possibly improved biomarkers for prostate cancer. In order to identify DNA methylation changes associated with aggressive prostate cancer, we performed a genome-wide analysis of 40 prostate cancers using Agilent human CpG island microarrays. Methylation profiles of candidate genes were validated using quantitative MethyLight technology in an independent series of 219 radical prostatectomies and compared to clinicopathological parameters. The effects of methylation on expression of HOXD3 and HOXD8 and the possible role of HOXD8 in progression of PCa were also investigated. We discovered previously unidentified methylation in the HOXD cluster of genes, namely HOXD3 and HOXD8, as well as TGFβ2 and GENE X as potential prognostic biomarkers. Furthermore, unsupervised clustering of samples by methylation signature indicated ERG oncogene expression as significantly different between clusters. Within the independent cohort, we observed strong correlations between Gleason score (GS) and HOXD3 as well as GENE X, while HOXD3 and HOXD8 methylation were associated with ERG expresson. TGFβ2 was an independent predictor of disease recurrence using Cox multivariate regression analysis. In gene expression studies, both HOXD3 and HOXD8 were elevated in cancers with poor prognosis, while DNA methylation did not correlate with expression levels. Both genes were found to contain alternative transcription start sites, explaining the poor correlation between methylation and expression. Finally, knockdown of HOXD8 expression did not have any effect on viable cells or cell motility in an in vitro model. These results indicate that a panel of novel DNA methylation markers distinguish indolent prostate cancers from aggressive ones, and that expression of HOXD3 and HOXD8 is regulated by mechanisms including, but not dependent on, DNA methylation.

High-grade serous ovarian cancer (HG-SOC) is the most lethal gynecologic malignancy. Although most respond to initial therapy, the vast majority of patients eventually recur and die of their disease. Understanding intra-tumor cellular heterogeneity and inter-patient variability is necessary to effectively cure HG-SOC. The work described in this thesis should help to speed the progress of ovarian cancer research in several ways. First, I generated a robust xenograft model that recapitulates the cellular heterogeneity of HG-SOC. In addition, I performed gene expression profiling on a subset of xenografts and showed that they recapitulate the inter-patient diversity of this disease. Second, I applied this model to pre-clinical testing of a folate-targeted imaging agent and showed that it can identify metastatic studding by PET/CT and fluorescence imaging. Using my xenograft model, I investigated the properties of tumor-initiating cells (TIC) and demonstrated that TIC in HG-SOC are rare. Furthermore, although CD133 marks most TIC, heterogeneity in the phenotype is observed within individual tumors and between different patients. Finally, I used a transformative technology, CyTOF, to develop a novel pipeline for prioritization of candidate TIC markers, as well as for characterization of cellular heterogeneity in primary HG-SOC samples.