Breast cancer remains the most common malignant disease in women worldwide. Despite the advantages of early detection and improved treatments, studies into the mechanisms that initiate and drive breast cancer progression are still required. Recent studies have identified RUNX1, which is an essential transcription factor for hematopoiesis, is one of the most frequently mutated genes in breast cancer patients. However, the role of RUNX1 in the mammary gland is understudied. In this dissertation, we examined the role of RUNX1 in both normal mammary epithelial and breast cancer cells. Our in vitro studies demonstrated that RUNX1 inhibits epithelial to mesenchymal transition (EMT), migration, and invasion, reflecting its tumor suppressor activity, which was confirmed in vivo. Moreover, RUNX1 also contributes significantly to inhibition of the phenotypes of breast cancer stem cells (CSC), which is responsible for metastasis and tumor relapse. We showed that Runx1 overexpression reduces the tumorsphere formation and cancer stem cell population. Overall, our studies provide mechanistic evidence for RUNX1 repression of EMT in mammary cells, anti-tumor activity in vivo and regulation of CSC-like properties in breast cancer. Our results highlight crucial roles for RUNX1 in preventing epithelial to mesenchymal transition and tumor progression in breast cancer. This RUNX1 mediated mechanism points to novel intervention strategies for early stage breast cancer.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20220号 / 医博第4179号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 野田 亮, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24125号 / 理博第4853号 / 新制||理||1694(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)准教授 板東 俊和, 教授 深井 周也, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Esophageal cancer (EC) is an aggressive form of cancer, including squamous cell carcinoma (ESCC) and adenocarcinoma (EAC) as two predominant histological subtypes. Accumulating evidence supports the existence of cancer stem cells (CSCs) able to initiate and maintain EAC or ESCC. In this review, we aim to collect the current evidence on CSCs in esophageal cancer, including the biomarkers/characterization strategies of CSCs, heterogeneity of CSCs, and the key signaling pathways (Wnt/β-catenin, Notch, Hedgehog, YAP, JAK/STAT3) in modulating CSCs during esophageal cancer progression. Exploring the molecular mechanisms of therapy resistance in EC highlights DNA damage response (DDR), metabolic reprogramming, epithelial mesenchymal transition (EMT), and the role of the crosstalk of CSCs and their niche in the tumor progression. According to these molecular findings, potential therapeutic implications of targeting esophageal CSCs may provide novel strategies for the clinical management of esophageal cancer.

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<p>Brain metastases are most common in adults suffering from lung cancer, predicting uniformly poor patient outcome and short survival time. Despite their frequency and severity, very little is known about the tumorigenesis of brain metastases. Previously developed primary brain tumour-initiating cell (BTIC) models were used to determine the presence of a stem-like population in brain metastases from the lung. Use of clinical samples and the NCI-H1915 cell line allowed for the development of useful strategies for study of brain metastasis.</p> <p>The sphere formation capacity and expression of known BTIC markers in brain metastases was suggestive of a self-renewing population. Differentiation studies demonstrated that neither clinical samples nor NCI-H1915 cells had neural lineage potential. Intracranial xenotransplant of clinical samples and NCI-H1915 cells into NOD-SCID mice led to formation of multiple focal masses throughout the ventricles; the tumours were also serially transplantable, further implicating a TIC population. Of known BTIC markers, only CD15 expression levels and patterns were similar enough in clinical samples and NCI-H1915 cells to warrant prospective sorting experiments in the cell line. Use of CD15 failed to identify a CSC or TIC population in NCI-H1915 cells.</p> <p>These findings suggest that a TIC population is present in brain metastases; however, this remains to be identified. It is recommended that due to the limitations of cell surface markers, the study of brain metastasis should use a selective gene expression approach, in order to target genes and pathways essential to metastasis. It was shown that NCI-H1915 cells could be useful for such an approach, studying the effects on proliferation, sphere formation, and tumour formation capacity of brain metastases from the lung. Further study using this model could ultimately lead to the disruption of pathways essential to the metastatic process, transforming a uniformly fatal disease into a more localized and treatable one.</p> / Master of Science (MSc)

<p>Based on the cancer stem cell (CSC) model, tumors develop as a hierarchy, much like normal tissue. At the apex, CSCs are capable of self-renewal and differentiating into non-tumorigenic cells that form the bulk of the tumor. CSCs have been isolated for many types of cancer based on cell surface marker expression. For instance in human breast carcinoma, the CD44+ CD24-/low EpCAM+ population are enriched in CSCs. These cells are more resistant to traditional chemo and radiation therapy relative to the bulk of the tumor. As such, many believe CSCs to be responsible for relapse and metastasis events. Hence, a more targeted therapy towards breast CSCs can prove to be very effective. EpCAM has been shown to be a reliable, albeit not exclusive, marker of breast CSCs. A more thorough understanding of EpCAM’s function can provide new angles for designing therapeutic agents. Originally thought to be a mere adhesion molecule, EpCAM is now known to derive a signalling pathway that promotes transcription in its target genes. We aimed to provide further insight into this novel pathway, by manipulating EpCAM’s expression and function in MCF7 breast cancer cell line. We designed a dominant negative allele of the EpCAM protein to hinder the activity of the endogenous EpCAM. However, this transgene offered no significant effect on our cell line. Furthermore, we used RNA interference technology to reduce EpCAM expression. The introduction of the EpCAM short hairpin RNA constructs into our cell line had inhibitory effects on transcription reporter expression, cellular growth, adherent colony and unattached sphere formation. The inhibition of EpCAM in MCF7 spheres was followed by signs of cell death, differentiation and an epithelial mesenchymal transition process. Sphere cultures are used because they are enriched in cancer stem cells. The adverse effects of EpCAM inhibition in MCF7 spheres provides further evidence as to the role of this protein in cancer stem cells.</p> / Master of Science (MSc)

Brain tumors represent a leading cause of cancer mortality, of which medulloblastoma (MB) and glioblastoma (GBM) represent the most frequent malignant pediatric and adult brain tumors, respectively. The identification of a rare clonal population of cells, termed cancer stem cells (CSCs) or brain tumor-initiating cells (BTICs), as having the ability to initiate, proliferate, and maintain tumor growth has offered a developmental framework for studying MB and GBM. Evidence in support of cell signaling programs carried forward from brain development into oncogenesis have provided opportunities for BTIC-directed therapies targeting the key BTIC property of self-renewal. Given that neural stem cells (NSCs) must maintain a relative balance between self-renewal and differentiation, brain tumorigenesis may be conceptualized as a disease of unregulated BTIC self-renewal. In this work, I aim to demonstrate the re-emergence of self-renewal genes that regulate NSCs in BTICs, use the Wnt pathway as a model by which these genes may be regulated in a context-specific manner, and identify clinically tractable therapies directed at the overall BTIC self-renewal signaling machinery. Specifically, in Chapter 2, I describe the presence of a shared signaling program between NSCs and MB BTICs consisting of Bmi1 and FoxG1. In Chapter 3, I provide evidence in support of a context-specific tumor suppressive function for activated Wnt/β-catenin signaling in MB. Lastly, in Chapter 4, I demonstrate a CD133-AKT-Wnt signaling axis in which CD133 functions as a putative cell surface receptor for AKT-dependent Wnt activation in GBM. Overall, the body of this thesis offers a mechanistic model by which BTICs may be regulated and targeted to impair tumor growth and improve overall survivorship in childhood MB and adult GBM. / Thesis / Doctor of Philosophy (PhD)