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)

Despite recent advancements in cancer treatment, the management of heterogeneous cancers poses a persistent challenge. This is best exemplified by the limited success in treating acute myeloid leukemia (AML), which still exhibits a 5-year survival rate of less than 30% to date. The resistance to treatment and high likelihood of relapse in AML patients is believed to be attributed to the elusive nature of cancer stem cells (CSCs). CSCs are characterized by their self-renewal capabilities and blockade to normal differentiation into mature hematopoietic cells, however they are only functionally defined through their engraftment potential upon transplantation into immunodeficient mice to phenocopy human tumorigenesis. Therefore isolation, study and treatment of CSCs is a prominent challenge in cancer research. To circumvent this challenge, our research group has developed a proprietary screening platform capable of identifying compounds within vast chemical space that selectively target surrogate human CSCs while preserving healthy stem cell counterparts. Our approach of screening and identifying compounds that selectively target human CSCs compared to normal SCs focuses on custom compound libraries curated through pre-screening for anti-proliferative and lethality properties in Escherichia coli (E. coli). From a pool of 21,000 active molecules selected from several libraries chemical space totalling 8.7 million compounds, we identified 15 distinct structural frameworks or "scaffolds", each representing different chemical structures that form the basis for potential drug development. Through secondary assays using primary AML patient derived cells, we have prioritized a novel compound, termed MLMB-2231 as a promising lead anti-CSC candidate. However, beyond MLMB-2231's demonstrated ability to selectively target human CSCs over normal stem cells, the cellular and molecular mechanisms of activity are unknown, limiting improvements and use towards investigational new drug application (IND) initiation. Here, I have utilized a variety of chemical genomics techniques to probe the downstream effects and gene targets of MLMB-2231. Apoptosis and cell cycling assays demonstrated that MLMB-2231 operates through an induction of apoptosis at 48h resulting in a G0/G1 cell cycle stall. The use of genome-wide CRISPR viability screening combined with transcriptomic analysis through RNA sequencing identified significant upregulation of pathways associated with aberrant ubiquitin- protease system (UPS), disruption of cell cycling, and upregulation of apoptotic pathways. These findings suggest that MLMB-2231 exerts its effects by interfering with the UPS, leading to impaired protein degradation and cellular stress, disrupting key cell cycle checkpoints, and inducing apoptosis. Future studies will focus on hit validation to confirm direct binding targets and incorporate animal models to evaluate pharmacokinetics (pK) and overall efficacy in vivo, paving the way for potential clinical applications. / 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)