Triple‐negative breast cancer (TNBC) is the most refractory subtype of breast cancer to current treatments and accounts disproportionately for the majority of breast cancer‐related deaths. Research has not yet identified specific therapies for TNBC and chemotherapy remains the conventional therapy in the clinic. While conventional chemotherapy regimens have demonstrated success at reducing bulk tumor burden, they have been shown to enrich cancer stem cells (CSCs). CSCs promote chemoresistance, metastasis, heterogeneous tumor regeneration and disease relapse. Owing to tumor plasticity and the conversion between CSC and non-CSC subpopulations development of a strategy capable of inhibiting both non-CSC and CSC subpopulations is crucial for TNBC therapy. In this compilation of my main research projects, several new approaches for the treatment of TNBC were identified which target not only the bulk tumor population but also the CSC populations residing within the tumor: 1. Co-suppression of Wnt, HDAC, and ESR1 using clinically relevant low‐dose inhibitors effectively repressed both bulk and CSC subpopulations and converted CSCs to non‐CSCs in TNBC cells. 2. Co-inhibition of mTORC1, HDAC, and ESR1 was capable of reducing both bulk and CSC subpopulations as well as the conversion of fractionated non-CSC to CSCs in in a human TNBC xenograft model and hampered tumorigenesis following treatment. 3. Inhibition of Wnt and YAP retarded tumor growth of TNBC cells in either epithelial or mesenchymal states, and both CD44high/CD24low and ALDH+ CSC subpopulations were diminished in a human xenograft model reducing tumorigenicity following treatment.

Thesis (M.S.)--Marshall University, 2008. / Title from document title page. Includes abstract. Document formatted into pages: contains 80 p. Includes bibliographical references (p. 60-67).

Tumour initiation and progression is thought to be driven by a small population of tumor initiating cells (TICs) or cancer stem cells (CSCs), which have the capacity to migrate and cause metastases and contribute to tumour relapse. These cells possess properties that are similar to those of normal tissue stem cells, which include the capacity to undergo self-renewal as well as the capacity to give rise to more differentiated progenitor cells, which comprise the bulk of the tumour cell population. Thus far, the clinical significance of these cells in breast cancers has not been extensively explored with regard to their relationship with tumour pathology or patient survival. In this thesis we evaluate the presence of these cells in terms of clinicopathological tumour characteristics and patient outcome, as well as assess potential markers of breast CSCs for prognostic significance. Through the quantification of breast CSCs in primary breast tumours using in vivo xenografts assays we show that their presence correlates with aggressive tumour characteristics. In addition, we propose that markers of breast CSCs may differ based on the molecular subtype of the tumour, and that these markers have prognostic significance in patients. / Thesis / Master of Science (MSc)

Melanoma is the most aggressive skin cancer with high recurrence and low survival rate. In addition to genetic mechanisms, resistance also arises from phenotypic heterogeneity in which a proportion of cells, the so-called melanoma stem or initiating cells, survive therapy. Due to a lack of reliable markers, however, there is still debate about the existence of these cells in melanoma. Consistent with phenotypic heterogeneity, previous observations in our laboratory have demonstrated that cells in melanoma can reversibly segregate in vivo into different subpopulations with different properties, such as differentiation or increased invasive capacity (potentially attributed to the existence of de-differentiated stem-like cells). To characterise these cells, a dual reporter lentiviral system was engineered, expressing fluorescent proteins under cell stage/phenotype-specific promoters. The promoters for the transcription factors POU3F2 (Brn2) (to mark de-differentiated cells) and the microphthalmia-associated transcription factor (MITF) (to mark proliferating and differentiated cells) were chosen. Lentivirally-transduced cells were used to screen a library of kinase inhibitors for their potential to affect promoter activity in vitro. The RhoA/ROCK pathway, known to contribute to invasion and metastases, was identified to play a role in Brn2 promoter activity and exhibited differential effects on both the MITF and Brn2 promoters in 501mel and SKmel28 cell lines. Through investigation of other signalling pathways involved in melanoma metastasis, we also identified the co-activator Mastermind-like 1 (MAML1), previously reported to act in the Notch pathway, as an activator of the Brn2 promoter via the transcription factor TCF3, and the MITF promoter through the lymphoid-enhancer binding factor 1 (LEF1). The effects of MAML1 on Brn2 and MITF promoter activity were potentiated by β-catenin. These findings provide new opportunities for the identification of therapeutic targets to prevent metastases formation in melanoma.

The cancer stem cell (CSC) theory proposes that a minor population in tumor cells with specific features, such as self-renewal and reproducible tumor phenotype could contribute to tumor relapse and chemotherapy resistance. Several studies have convincingly documented the existence of ovarian CSC, but questions related to the biologic behavior and specific biomarkers of ovarian CSC remain to be clarified. In the present study, we firstly established a tumor cell line with capability of regenerating tumors through serial transplantation of ovarian tumor tissue in non-obese/severe combined immunodeficient (SCID) mice. After separation of CD133+ cells with magnetic beads, we compared the phenotype and biologic behavior of CD133+ versus CD133-cells. It was found that the CD133+ cells were much more potent to produce colonies in semi-solid agar culture than CD133-cells. The proportion of the cells in G0/1 cell cycle is much higher in CD133+ cells than in CD133-cells. Furthermore, in vivo experiments demonstrated that the CD133+ cells were capable of repeatedly regenerate tumors in NOD/SCID mice, while the CD133-cells were not. Compared with CD133-cells, the CD133+ cells expressed much higher levels of the stem cell markers Oct4, Sox2, Nanog and Mcl-1. Clinically, among a total of 290 ovarian epithelial cancers, increased level of CD133 expression was positively correlated with a high cancer stage and had a worse 5-year survival rate. Taken together, the results suggest that the CD133+ cells from human ovarian cancer have the characteristics of CSC, which may contribute to ovarian cancer relapse and anti-apoptotic activity. The method of ovarian CSC enrichment we established provides a feasible and practical way of ovarian cancer research in a molecular level. In addition, CD133 may be used as a prognostic marker for ovarian epithelial cancer, which may have a role for future therapeutic effect.

Cancer is a highly complex disease that evolved in response to a wide range of biological and molecular changes that impact disease behaviour, treatment efficacy and clinical outcomes. Studying this diversity in human tumours is essential for gaining insights that will ultimately improve the survival rates of cancer patients. Cancer stem-like cells (CSCs) are believed to be responsible for invasive and metastatic features in tumours and can contribute to chemotherapy resistance and subsequent tumour relapses. There is an increasing need to identify the molecular mechanisms involved in tumour cells, particularly in CSCs. Cancer testis antigens (CTAs) are a subclass of germline proteins normally produced in immune-privileged sites, such as the testis, ovary and placenta of somatic tissues, and the presence of these antigens is increased in a variety of cancers. These characteristics make CTAs highly important immunotherapeutic targets, since they do not harness the immune response in the testes but encode immunogenic proteins that can induce a specific response in cancerous tissues. CTA genes are potentially very importance in clinical applications, including cancer diagnosis, vaccination and immunotherapy. This current study focused on the investigation of two CTAs, TDRD12 and LKAAEAR1, that may have an enhanced presence in cancer and the potential to be immunogenic. TDRD12 is linked to stemness features and enables the proliferation of germ line tumour cells. It appears to act as a possible transcriptional regulator for germline factors that are essential to cell cycle proliferation, germ cell maintenance and stem marker expression. TDRD12 may have the potential to drive oncogenesis and CSC targets. LKAAEAR1 was validated as a CTA at the protein level, showing its production was restricted to germ cells and the central nervous system from normal tissues and showed aberrant production in a wide range of tumours. This protein has been shown to be produced in germ cells undergoing spermatogenesis with strong nuclei staining, suggesting its potential role in this process. LKAAEAR1 potentially acts as a regulator for transposable elements, thereby increasing its contributions to cancer development. This study demonstrated that LKAAEAR1 could potentially be used as a cancer biomarker and therapeutic target.

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.

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.

On average, 65 Canadian men will be diagnosed with prostate cancer (PC) every day, making it the most common male cancer in Canada. Despite the prevalence, the etiology of PC is unknown. Evidence nonetheless supports the role of prostate cancer stem cells (PCSCs) in PC initiation and metastasis. In spite of almost a decade worth of research on PCSCs our knowledge on their biology remains fragmented. By taking advantage of the availability of DU145 cell-derived PCSCs in our laboratory, this thesis research focuses on investigating the unique properties of PCSCs and their function in promoting PC tumorigenesis. We identified two PCSC-specific proteins, ALDH3A1 and CNTN1. In mouse models of xenograft tumors, ALDH3A1 was expressed at higher levels in PCSC-derived tumors than in DU145 non-PCSC-produced tumors and in lung metastases than local tumors. In clinical settings, elevation of ALDH3A1expression was observed from normal prostate tissues to carcinomas and from local PCs to the paired lymph node metastases. Additionally, ALDH3A1 was clearly detected in bone metastases. Similar to ALDH3A1, CNTN1 expression associates with PC progression and biochemical recurrence following radical prostatectomy. The clear presence of CNTN1 in lymph node and bone metastases was also demonstrated. Furthermore, CNTN1 expression promoted PC metastasis to the lungs and tumor initiation in NOD/SCID mice. Mechanistically, CNTN1 increased AKT activation and reduced E-cadherin expression. Collectively, our research revealed important roles of both PCSC proteins in promoting PC tumorigenesis and progression. PC develops chemotherapy resistance in which PCSCs play a major role. In supporting this knowledge, we demonstrated that PCSCs are innately more resistant to the chemotherapeutic drugs, etoposide and docetaxel and that this resistance was in part attributable to their enhanced DNA damage response. Taken together, the findings of this thesis advances our knowledge on two specific PCSC markers and their association with prostate cancer progression and metastasis. As well as to the mechanism whereby PCSCs promote resistance to chemotherapeutic drugs. / Thesis / Doctor of Philosophy (PhD)

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Out of the four molecular subgroups (WNT, SHH, Group 3 and Group 4), Group 3 patients face the highest incidence of leptomeningeal spread and overall patient survival of less than 50%. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naïve tumors, provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumors. The paucity of patient matched primary and recurrent MB samples has contributed to the lack of molecular targets specific to medulloblastoma recurrence, limiting relapsing MB patients to palliation. Our previous in silico analyses revealed enriched expression of many stem cell self-renewal regulatory genes in Group 3 MB. In this work, I have set out to investigate whether by identifying genes contributing to self-renewal of Group 3 MB cells, we can characterize a population of cells responsible for therapy evasion and subsequent tumor relapse. Initially, we have adapted the existing COG (Children’s Oncology Group) protocol for children with newly diagnosed high-risk MB for treatment of immuno-deficient mice intracranially xenografted with human MB cells. Cell populations recovered separately from brains and spines mice during the course of tumor development and therapy were comprehensively profiled for gene expression analysis, stem cell and molecular features to generate a global, comparative profile of MB cells through therapy. Additionally, we have investigated therapeutic potential of small molecules targeting BMI1, a known self-renewal regulating gene. In the setting of recurrent Group 3 MB, pharmacological inhibition of BMI1, led to a remarkable decrease in cell proliferation and self-renewal in vitro as well as reduction of local and spinal metastatic disease in vivo. Finally, by combining the established therapy-adapted patient-derived xenograft mouse model and BMI1 inhibitor, PTC-596, we have demonstrated an additive effect of two modalities and provided the pre-clinical data for the upcoming Phase I trial. Biological investigations into the drivers of MB recurrence will lead to development of new therapeutic options for children who are frequently limited to palliation. Clinically relevant mouse models of MB recurrence can serve as platforms for pre-clinical testing and validation of new treatments aimed to provide therapeutic intervention rather than palliation. / Thesis / Doctor of Philosophy (PhD) / Medulloblastoma is the most common type of brain cancer that affects children. Out of the four main subgroups of medulloblastoma, tumors in Groups 3 and 4 are the most aggressive and are associated with a low overall survival in children diagnosed with this type of brain cancer. These two subtypes of medulloblastoma also account for the largest number of patients in which gold standard therapies fail and no additional therapies are available. Several studies have shown the existence of few cells within the tumor that alone can drive tumor growth. The aggressive behavior of these cells has in part been attributed to dysregulation of genes involved in cell replication and division. Further studies that will focus on understanding the significance of genes that regulate cell growth and replication can help discover a population of cells that is capable of evading therapy and contribute to tumor relapse. The identification and characterization of such population can lead to development of novel treatments for the children affected with aggressive medulloblastoma. In my thesis, I have developed a mouse model that replicates the aggressive therapy given to the medulloblastoma patients in order to study cells capable of escaping the harsh treatment and drive tumor comeback. Next, by profiling the gene expression and functional attributes of those cells, we identified genes that contribute to regulation of cell division and growth. The effects of both increasing and decreasing the activity of those genes were then tested in cells grown in the dish. Subsequently, the most promising results were verified in the established mouse models. The main objective of my thesis was to discover new opportunities in treatments the most aggressive type of brain cancer affecting children, and thus not only improve the quality of treatment but also the overall survival of patients with medulloblastoma.