Introduction: Objectives were to describe time intervals between presentation to a tertiary care center, diagnosis and treatment in pediatric acute lymphoblastic leukemia (ALL) and Hodgkin disease (HD), and measure their impact on overall survival (OS) and event-free survival (EFS). Methods: Children in POGONIS (Pediatric Oncology group of Ontario Networked Information System) with ALL or HD from 1997-2007 were eligible. Time intervals were dichotomized at clinically defined cut-points. OS and EFS were examined with univariate and multivariable Cox proportional hazards (CPH) models. Results: In ALL, in multivariable analysis, those with treatment > 3 days after diagnosis had inferior OS (adjHR=2.49; 95%CI 1.4-4.43;p=0.002), and inferior EFS (adjHR=1.73; 95%CI 1.01-2.96;p=0.047). In HD, in multivariable analysis, those with treatment > 7 days after diagnosis had superior EFS (adjHR=0.37; 95%CI 0.18-0.77;p=0.008). Conclusions: Time to treatment is associated with survival in ALL and HD. Future research will further delineate the relationship between time to treatment and outcome.

BACKGROUND: The incidence of liver metastases from colorectal cancer (CLM) is on the rise. Older cancer patients are frequently subject to under-treatment. METHODS: A Markov decision model was built to examine the effect on life expectancy (LE) and quality-adjusted life expectancy (QALE) of four strategies – best supportive care (BSC), systemic chemotherapy (SC), radiofrequency ablation (RFA), and hepatic resection (HR). The model was designed to account for both age and comorbidities. RESULTS: In the base case analysis, BSC, SC, RFA, and HR yielded LEs of 11.9, 23.1, 34.8, and 37.0 months, respectively, and QALEs of 7.8, 13.2, 22.0, and 25.0 months, respectively. Model results were sensitive to several variables including age, comorbidity status, and length of model simulation. CONCLUSION: Hepatic resection may be the optimal treatment strategy for healthy older patients with CLM. Treatment decisions in older cancer patients should be individualized and account for patient age, comorbidities, and values.

The pediatric sarcoma alveolar rhabdomyosarcoma (ARMS) is highly aggressive with a poor prognosis for diagnosed patients. Here, we demonstrate that targeting the unique oncogene integrin-linked kinase (ILK) in ARMS cells in conjunction with the common chemotherapy agent vincristine, a synergistic effect is found in the reduction of cell viability in vitro. This result was achieved by both RNAi-mediated depletion of ILK and using a small molecule kinase inhibitor specific for ILK. Both techniques were found to disrupt important protein interactions at the site of the centrosome. Combination ILK disruption and vincristine treatment of cells induced the expression of apoptotic markers and arrested cells in the G2/M stage of the cell cycle. Interestingly, protein levels of JNK and its target c-Jun were regulated with combined treatment. Altogether, these findings indicate that the use of molecular targets like ILK may further improve the clinical treatment of ARMS.

Polo-like kinase 4 (Plk4) has been identified as a molecular marker of resistance to therapy in cancer. Our laboratory has recently shown a motility defect in Plk4+/- murine embryonic fibroblasts (MEFs) compared to Plk4+/+. I hypothesized that Plk4 augments cancer cell motility. Plk4 depletion with siRNA in Plk4+/+ MEFs and HeLa cells suppressed invasion compared to control. Transient over-expression of Flag-Plk4 in cancer cell lines did not consistently increase invasion. However, modest Plk4 over-expression using stable clones showed higher invasion rates than non-induced. The RhoGEF Ect2, an important Plk4 substrate, transiently localized to protrusions in MEFs, suggesting a RhoA-based signalling cascade in motility. The effect of Plk4 heterozygosity on metastasis was tested in a transgenic mouse model but there was no significant difference in developing metastasis compared to wild type. Further studies are required to characterize the effect of Plk4 on motility, and its potential as a therapeutic cancer target.

In Canada, ovarian cancer kills about 67% of diagnosed patients, largely due to difficulties in early diagnosis. Current treatment consists of debulking surgery and intermittent chemotherapy every three weeks. This approach leads to insufficient drug concentrations at disease sites, and long treatment-free intervals cause accelerated tumor proliferation and drug resistance, resulting in a 5-year survival rate of only 25-35%. Drug resistance development is the ultimate cause of the majority of patient deaths. Improvements yielding more effective treatment are fundamental for successful management of this disease. This thesis investigated a continuous chemotherapy strategy devoid of treatment-free intervals for ovarian cancer treatment. A biocompatible, biodegradable polymer-lipid injectable formulation PoLigel, was used for continuous DTX delivery. The formulation was well tolerated; no alterations in body weight, behaviour, histology of peritoneal tissues, or interleukin-6 levels were seen in CD-1 mice treated with the PoLigel. Continuous DTX therapy via the PoLigel was considerably more efficacious than intermittent therapy, resulting in significantly less tumor burden and ascites fluid in models of human and murine ovarian cancer. Continuous therapy resulted in less tumor cell proliferation and angiogenesis, and more tumor cell death than intermittent DTX. The presence and length of treatment-free intervals was shown to contribute to the development of drug resistance. Eliminating these intervals by continuous dosing resulted in superior antitumor efficacy in both chemosensitive and chemoresistant xenograft models of human ovarian cancer, and prevented drug resistance increase after a 21-day treatment period. Survival studies revealed that intermittent dosing led to a mild survival prolongation of 36% and 10% in chemosensitive and chemoresistant models, respectively, whereas continuous DTX prolonged survival by a striking 114% and 95%. Although long-term continuous chemotherapy substantially improved survival, increased drug resistance mechanisms were found at the endpoint. Overall, results presented here encourage the clinical implementation of continuous chemotherapy due to greater achievable therapeutic advantages.

MicroRNA is a single-stranded RNA molecule of about 22 nucleotides in length and is expressed endogenously. It functions as a gene regulator by pairing imperfectly with 3’ untranslated region (3’UTR) of target mRNAs, leading to translational inhibition. MicroRNA is implicated in many regulatory pathways and hence affects various cellular activities. In the development of cancer, genetic alterations occurred at miRNA locus and its expression level is dysregulated in various cancers versus normal tissue counterparts. It is thus important to find the targets of dysregulated microRNAs contributing to progression of cancer. To facilitate long term functional studies, a microRNA expression construct with unique futures was generated. Stable expression of miR-378 enhanced cell survival, reduced caspase-3 activity, and promoted tumor growth and angiogenesis. By algorithmic predictions and proteomic analysis, two tumor suppressors, SuFu and Fus-1, were found to be translationally regulated by miR-378. Target validation was confirmed by co-transfection experiments and luciferase activity assays, reassuring its oncogenic role by regulating two tumor suppressor genes simultaneously. Conversely, microRNA can also function as a tumor suppressor by modulating expression of Versican, an extracellular matrix protein known to facilitate tumorigenesis and angiogenesis. By a novel PCR method, more than one microRNA were found to bind to Versican 3’UTR. iii Among these microRNAs, targeting of Versican and Fibronectin by miR199a-3p was validated. Expression of a fragment of Versican 3’UTR was expected to antagonize the function of miR-199a-3p. Stable expression of Versican 3’UTR resulted change in cell morphology and increased cell-cell adhesion. Analysis of primary tissues from transgenic mice expressing versican 3’UTR showed an increase expression of Versican and Fibronectin, and organ adhesion was found between liver and its surrounding tissues. In addition, 3’UTR also modulated the level of miR-199a-3p and miR-136, alleviating translation of negative cell cycle regulators, PTEN and Rb1. This resulted in reduced cell proliferation and hence diminished tumor growth. These findings suggest a role of microRNA in tumor growth, providing a valuable target for therapeutic intervention.

Chemotherapy is often administered once every three weeks to allow repopulation of essential normal tissues such as the bone marrow. Repopulation of surviving tumour cells can also occur between courses of chemotherapy and can decrease the efficacy of anticancer treatment. This thesis aims to characterize repopulation, to study the effect of targeted cytostatic agents to inhibit repopulation, and to determine the optimal scheduling of chemotherapy and molecular targeted treatment. The distribution of proliferating and apoptotic cells in human squamous cell carcinoma (A431) xenografts was studied following chemotherapy using fluorescence immunohistochemistry. There was an initial decrease in cell proliferation and in the total functional blood vessels, and an increase in apoptosis observed following treatment with paclitaxel chemotherapy. A rebound in cell proliferation occurred approximately 12 days following treatment, which corresponded with a rebound in vascular perfusion. The effect of gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, to inhibit repopulation between courses of chemotherapy was determined using EGFR-overexpressing A431 cells and xenografts. Furthermore, concurrent and sequential schedules of combined chemotherapy and molecular targeted treatment were compared. Gefitinib inhibited the repopulation of A431 cells in culture when administered sequentially between chemotherapy; sequential treatment was more efficacious than concurrent treatment probably because concomitant scheduling rendered quiescent cells less responsive to chemotherapy. However, in vivo studies using chemotherapy in combination with gefitinib or temsirolimus, a mammalian target of rapamycin (mTOR) inhibitor, showed that concurrent scheduling of combined treatment was more effective at delaying regrowth of xenografts than sequential treatment; this was likely due to dominant effects on the tumour microenvironment. The work completed in this thesis has shown that repopulation occurs in A431 xenografts following paclitaxel treatment, and these changes are associated with changes in the tumour vasculature. Repopulation of A431 cells was inhibited by gefitinib administered sequentially with paclitaxel. However, studies in mice showed better inhibitory effects when chemotherapy was given concomitantly with cytostatic agents such as gefitinib or temsirolimus. Our in vivo data highlight the importance of characterizing changes in the tumour microenvironment when determining optimal scheduling of chemotherapy and molecular targeted treatment.

FDA-approved drugs with previously unrecognized anti-cancer activity could be rapidly repurposed for this new indication. We compiled a library of such off-patent drugs to screen four leukemia cell lines and identified the anti-parasitic agent ivermectin that induced cell death at low micromolar concentrations. In cell death and clonogenic growth assays, low micromolar concentration of ivermectin significantly reduced viability of leukemia cell lines and patient samples compared to normal peripheral blood stem cells. In xenograft mouse models of leukemia, ivermectin decreased tumor volume and weight by up to 72% when compared to control without observable toxicity at pharmacologically achievable dosage. In this study, we further demonstrate that ivermectin activates chloride channels in leukemia cells leading to membrane hyperpolarization and increased reactive oxygen species generation. In addition, it demonstrated synergistic interaction when used in combination with Daunorubicin and Cytarabine. Therefore, this study highlights a potential new therapeutic strategy in repurposing ivermectin for the treatment of AML.

Glioblastoma (GBM), similar to many other cancers, exhibits enhanced aerobic glycolysis with concomitant lactate production, a phenomenon known as the Warburg effect. We have demonstrated that preferential expression of Hexokinase 2 (HK2) is a critical mediator of metabolic reprograming in GBMs and its inhibition is a potential therapeutic strategy for sensitization of GBM tumors to radiation (RAD) and/or temozolomide (TMZ). Our results indicate that conditional HK2 inhibition disrupts energy homeostasis and sensitizes GBMs to radiochemotherapy under hypoxia. In GBM xenografts, conditional HK2 loss sensitizes GBM tumors to concomitant RAD/TMZ and results in a significant survival benefit in the mice. Moreover, loss of HK2 resulted in GBM remodeling with HK2 knockdowns showing increased necrosis, hypoxia, inflammatory infiltration and reduced vascularization. We anticipate that targeting a key metabolic enzyme involved in the Warburg effect might improve the efficacy of current therapeutic regimen and provide a unique paradigm for the management of GBMs.

Glioblastoma (GBM), similar to many other cancers, exhibits enhanced aerobic glycolysis with concomitant lactate production, a phenomenon known as the Warburg effect. We have demonstrated that preferential expression of Hexokinase 2 (HK2) is a critical mediator of metabolic reprograming in GBMs and its inhibition is a potential therapeutic strategy for sensitization of GBM tumors to radiation (RAD) and/or temozolomide (TMZ). Our results indicate that conditional HK2 inhibition disrupts energy homeostasis and sensitizes GBMs to radiochemotherapy under hypoxia. In GBM xenografts, conditional HK2 loss sensitizes GBM tumors to concomitant RAD/TMZ and results in a significant survival benefit in the mice. Moreover, loss of HK2 resulted in GBM remodeling with HK2 knockdowns showing increased necrosis, hypoxia, inflammatory infiltration and reduced vascularization. We anticipate that targeting a key metabolic enzyme involved in the Warburg effect might improve the efficacy of current therapeutic regimen and provide a unique paradigm for the management of GBMs.