It is well established that the Wnt pathway is associated with tumorigenesis in a wide range of human cancers, including a majority of breast cancers. However, due to diverse roles of Wnt signalling, therapeutic targeting has not yielded consistent results and underlying mechanisms remain unclear. Here, I show that breast cancer cell lines with high E-cadherin expression are resistant to TCF4 inhibitors and develop cancer stem cell characteristics. Conversely, cells with low levels of E-cadherin are very susceptible to cell death with the same treatment. My results suggest that breast cancer cells in an epithelial-like state, but not mesenchymal-like state, will be more responsive to therapeutic targeting of the Wnt/TCF pathway. Importantly, E-cadherin high cells show robust Akt activation, whereas E-cadherin low cells do not. Thus, combinational inhibition of both Wnt and Akt signalling is needed to effectively target breast cancer cells in both the epithelial and mesenchymal states.

The incidence of melanoma and non‐melanoma skin cancer is continuing to increase worldwide. Melanoma is the sixth most common cancer in the United States, making skin cancer a significant public health issue. Photo chemoprevention with natural products is an effective strategy for the control of cutaneous neoplastic. Polyphenols from fruits have been shown to protect the skin from the adverse effects of solar UVR, cancer, and the growth of cancer stem cells. In particular, blueberries are known for their high concentration of phenolic compounds that have the high antioxidant capacity, and their effectiveness in reducing UV damage and, therefore, skin cancer. In Matar's lab, we have shown that Polyphenol-Enriched Blueberry Preparation (PEBP), derived from biotransformation of blueberry juice through fermentation, is effective for targeting skin cancer stem cell proliferation in different skin cancer cell lines. We predicted that PEBP affects melanoma skin cancer stem cells (MCSCs) epigenetically by targeting miRNA pathways. We observed the effects of PEBP on sphere growth and cell motility in vitro. We performed RT2-qPCR analyses to determine PEBP influence on miRNA in B16F10 spheres. We transfected B16F10 cells with miR-200b and performed western blotting analyses. Our results demonstrated that PEBP reduced sphere growth and cell migration, and up regulated miR-200b expression in different biological settings. Inhibition of miR-200b increased Zinc Finger E-Box Binding Homeobox 1 (ZEB1) expression. Consequently, PEBP may influence MCSCs through miRNA pathways. Elucidating the mechanisms by which PEBP modulates CSCs biological behavior by controlling miRNAs will enhance our understanding of the molecular mechanisms in skin cancer chemoprevention and might result in their use as natural photo-protectants in skin cancer.

Increasing evidence suggests that breast cancer is caused by cancer stem cells and the cure of breast cancer requires eradication of breast cancer stem cells. In this study, we established and characterized a sphere culture model derived from side population cells from the human breast cancer cell line MCF7. The sphere culture could be maintained long term and was enriched in cells expressing known breast cancer stem cell marker CD44+CD24 -. These sphere cells showed higher colony formation ability in vitro and higher tumorigenicity in vivo than MCF7 cells, suggesting the enrichment of breast cancer stem/progenitor cells. To identify compounds that preferentially inhibit the sphere cells, we performed a compound library screening. Two lead compounds, NSC24076 and NSC125034 and an analog of NSC125034, 8-quinolinol (8Q), were identified as having preferential activity against the sphere cells. 8Q showed some antitumor activity alone but had much better therapeutic effect and relapse prevention when combined with paclitaxel than either 8Q or paclitaxel alone in both MCF7 and MDA-MB-435 xenograft models. We propose that compounds selectively targeting cancer stem/progenitor cells when combined with standard chemotherapy drugs may produce an improved treatment of cancer without significant relapse.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24804号 / 医博第4996号 / 新制||医||1067(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 武藤 学, 教授 藤田 恭之, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Ovarian cancer is known as the "silent killer," due to its late diagnosis and frequent recurrence after initial treatment.  Finding a new way to diagnose and treat ovarian cancer in conjunction with current therapies is paramount.  By capitalizing on metabolic changes that occur during cancer progression, interventions can be developed.  The Nobel laureate Otto Warburg is credited with discovering an altered metabolic state within cancer cells known as the Warburg effect.  In the Warburg effect, cancer cells participate in an increased rate of aerobic glycolysis with an excess secretion of lactate, allowing for carbon flux into biosynthetic pathways.  Exactly which metabolic pathways are altered in ovarian cancer and at which stage in the progression of ovarian cancer they are occurring was unknown.  Therefore using the recently established mouse ovarian surface epithelial (MOSE) progression model, we were able to measure metabolic changes in varying states of disease and levels of aggressiveness.  As cells progressed from a benign early stage (MOSE-E), through a transitional intermediate stage (MOSE-I), to an aggressive late stage (MOSE-L), the MOSE cells became more glycolytic and lipogenic, establishing the MOSE model as a valuable model for studying ovarian cancer metabolism.  Treating the MOSE cells with the naturally occurring chemotherapeutic agent sphingosine decreased p-AKT  protein levels in the cell, decreased the glycolytic rate and decreased de novo cholesterol synthesis.  Cancer stem cells are known to be resistant to chemotherapy treatments and targeting their metabolism may be promising for combinatorial treatments.  Therefore, the metabolism of highly aggressive tumor-initiating cells (TIC), harvested from ascites of C57Bl/6 mice injected with MOSE-L cells were characterized.  Although the basal metabolism of the TICs was similar to the MOSE-L cells, TICs were more resistant to cell death as a consequence of external stresses and substrate depletion.  The TICs could also up-regulate oxygen consumption rate (OCR) when uncoupled and increase glycolysis when ATP Synthase was inhibited, highlighting their resiliency.  Taken together, we have identified targets for treatment strategies that could suppress the growth of primary tumors and may be effective against TICs, thereby suppressing tumor recurrence and possibly prolonging the life of women with ovarian cancer. / Ph. D.

Yes / Glioblastoma multiforme (GBM) is a grade IV brain tumor characterized by a heterogeneous population of cells that are highly infiltrative, angiogenic and resistant to chemotherapy. The current standard of care, comprised of surgical resection followed by radiation and the chemotherapeutic agent temozolomide, only provides patients with a 12–14 month survival period post-diagnosis. Long-term survival for GBM patients remains uncommon as cells with intrinsic or acquired resistance to treatment repopulate the tumor. In this review we will describe the mechanisms of resistance, and how they may be overcome to improve the survival of GBM patients by implementing novel chemotherapy drugs, new drug combinations and new approaches relating to DNA damage, angiogenesis and autophagy.

In the United States, metastatic breast cancer kills approximately 40,000 women and 400 men annually, and approximately 200,000 new cases of breast cancer are diagnosed each year. Worldwide, breast cancer is the leading cause of cancer deaths among women. Despite advances in the detection and treatment of metastatic breast cancer, mortality rates from this disease remain high because the fact is that once metastatic, it is virtually incurable. It is widely accepted that a major reason breast cancer continues to exhibit recurrence after remission is that current therapies are insufficient for targeting and eliminating therapy-resistant cancer cells. Emerging research has demonstrated that these therapy-resistant cells possess stem cell-like properties and are therefore commonly referred to as breast cancer stem cells (BCSCs). A major hallmark of BCSCs is the cell surface expression of CD44 and lack of expression of CD24, the so-called CD24-/CD44+ phenotype. Research indicates that this dangerous and rare subpopulation of BCSCs may be responsible for cancer onset, recurrence, and ultimately metastasis that leads to death. Two different model systems were utilized in this research. The first was the MCF7 cell line, a luminal A tumor subtype representative of a mildly invasive breast ductal carcinoma with an ER+/PR+/-/HER2- immunoprofile. The second was the MCF10A breast cancer progression model, which consists of three cell lines: MCF10A, MCF10AT1, and MCF10CA1a. In this system, spontaneously immortalized, non-malignant MCF10A cells were transfected with constitutively active H-Ras to form pre-malignant MCF10AT1 cells, which were then subcutaneously injected into mice and allowed to metastasize in order to form the oncogenic MCF10ACA1a cell line. This thesis presents evidence of a CD24low/-/CD44+ BCSC subpopulation within the MCF10A breast cancer progression model system. Findings indicate that RUNX1 and RUNX2 expression levels are involved in maintaining the BCSC phenotype. Across two different model systems, qRT-PCR analysis revealed that decreased levels of RUNX1 expression and increased levels of RUNX2 expression are essential for the maintenance of the BCSC subpopulation. It was also shown that low expression levels of RUNX1 and high expression levels of RUNX2 are present in CD24low/-/CD44+ BCSCs as compared to CD24+/CD44+ non-BCSCs. Furthermore, shRNA knockdown of RUNX1 was shown to enhance tumorigenicity, while shRNA knockdown of RUNX2 repressed tumorigenicity in BCSCs, as measured by the tumorsphere-formation assay. This research lays the groundwork for future investigations into the roles of RUNX1 and RUNX2 in regulating stemness in breast cancer.

Adult stem cells are a small population present within several tissues of an individual, possessing two unique properties: one, the ability to differentiate to give rise to all the cell types of the tissue, and second, the ability to self-renew and make more of their own kind. Owing to these two properties, stem cells underlie the process of organogenesis during development and tissue homeostasis in adult life. In the past decade a small sub-population of cells having phenotypic and functional properties similar to normal stem cells have been identified within several tumors. Only this sub-population of cancer cells seems to have the ability to both initiate and maintain tumors. These cells have been termed as ‘cancer stem cells’ (CSCs) owing to their striking similarities with the normal stem cells of the tissue. It is therefore of fundamental importance to understand normal stem cell biology in order to understand tumorigenesis. The rarity of normal stem cells within adult tissues, the absence of specific cell surface markers to identify and isolate them, and the absence of suitable culture conditions to maintain them has marred our understanding of stem cell behaviour. Recently, growth of mammary cells in serum free suspension cultures resulted in the generation of floating spheroids termed “mammospheres” that were shown to be enriched in stem/progenitor cell population. We established the mammosphere system in our laboratory using mastectomy samples obtained from the Kidwai Memorial Institute of Oncology. In order to understand the composition of the spheres, the stem cell characteristics within them, and the long term self renewal potential of human mammary epithelial stem cells, a detailed phenotypic and functional characterization of the mammospheres was carried out. Phenotypic Characterization: Confocal microscopy of propidium iodide stained mammospheres demonstrated that these spheres are cellular and not hollow structures. Immunostaining revealed that primary mammospheres expressed the epithelial markers like E Cadherin, ESA, CK14, CK18 and CK 19, but failed to express nestin or CD34, indicating their epithelial origin, devoid of contamination from haematopoeitic or neural stem cells. The sizes of mammospheres ranged from 40 to 110 μm, while that of the cells within them ranged from 9-15 μm. Although the sizes of the largest and smallest spheres through subsequent passages remained consistent, the proportion of small spheres increased in later passages. These results indicate the difference in the sphere initiating cells. While a large sphere might be generated by a stem cell, a smaller sphere might be originating from a progenitor. Thus, heterogeneity exists within mammospheres, with respect to size and composition. Unique cell surface markers coupled with flow cytometry serves as useful tools to isolate stem cells. However, no specific marker profile has been reported for normal human breast stem cells. In several tissues, like blood, brain etc, markers of normal stem cells have been successfully used to isolate cancer stem cells within that tissue. Since breast cancer stem cells have already been identified as CD24low/-44high cells, we explored if the same marker profile would hold true to identify normal breast stem cells as well. Two-colour based flow cytometry revealed that only the CD24low/-44high subpopulation of mammospheres could re-generate mammospheres, as well as give rise to all the other cellular fractions. These data demonstrated that normal and cancerous breast stem cells share identical marker profile. Functional Characterization: In addition to cell surface markers, a Hoechst dye based strategy used to isolate stem cells, exploits their unique property to efflux certain lipophilic drugs and small molecules due to the overexpression of ABC family of cell surface transporters. Cells effluxing Hoechst appear as a low fluorescing ‘Side population’ (SP) in a bivariate FACS plot. We detected a small, but distinct SP in human breast cells, which had a CD24low44low profile, and failed to initiate new mammospheres. Thus, the SP cells in mammospheres failed to correspond to the stem cell subpopulation. The hallmark feature of a stem cell is its long term self renewal ability, given that it is the longest lived cell in the body. Long term culture of mammospheres was carried out by passaging the spheres every week. We failed to observe mammosphere formation beyond four passages though there were live, proliferating and undifferentiated cells in fourth passage spheres. These results suggested that either the mammopsheres didn’t contain stem cells to begin with, or their stemness is restricted to four in vitro passages. In order to assess if mammospheres contained stem cells to begin with, we assayed for telomerase activity, since in the adult tissue, only stem cells retain telomerase activity. Telomerase, an enzyme that maintains the length of telomeres through multiple rounds of cell division, is not active in somatic cells. We detected the expression and activity of this enzyme in primary mammospheres, suggesting that the spheres may contain stem cells withinthem Another unique property of a stem cell is its ‘quiescence’, owing to their infrequent divisions. This property is studied by chasing a label (like BrdU or H3-Thymidine), which is taken up by the cells at an earlier time point and retained within the cell after prolonged periods, like weeks or months. In long term culture of mammospheres, using BrdU as the label, 1-2 distinct cells could be detected within late passage spheres which had retained the label, indicating that stem cells may be present within the fourth passage mammospheres as well. Staining for β-Galactosidase activity revealed that almost 70% cells derived from fourth passage spheres were senescent. We speculated that this senescent environment might be one of the inhibitory reasons for further mammosphere formation. Alteration of mammosphere culture conditions for long term maintenance of stem cells. A high level of atmospheric O2 is known to be one of the reasons for inducing senescence in cells. Culturing cells in conventional tissue culture conditions exposes them to high levels of O2 (21%) as against the physiological levels of 1-3% O2. Therefore, to assess the effects of lowered, or physiologically relevant levels of O2 on mammosphere stem cell biology, the mammospheres were cultured in 3% O2. Under this altered condition, a close to 3-fold increase was observed in the number of mammospheres formed coupled with a significant increase in their survival and proliferation. In order to understand the molecular basis of this observation, a microarray based global gene expression profiling was carried out. We observed a significant upregulation of VEGF, a gene responsive to hypoxia; three growth factor related genes, namely adrenomedullin, cMET and osteopontin. Upregulation of β Catenin, the downstream effector of the Wnt signaling pathway was also observed, indicating a possible mechanism for the increase in self renewal seen in 3% O2. We also observed downregulation of the cell cycle inhibitor, Chk1, which in part might explain the observed increase in proliferation. The increase in the number of proliferating cells might be one of the reasons for an increase in the number of spheres, as observed in 3% O2. Even though a significant decrease in the number of senescent cells was detected at 3% O2, mammosphere formation was not seen beyond four passages. It is therefore possible that there are other physico-chemical parameters, comprising the niche of the mammospheres, coupled to the O2 level, which need to be improvised for long term culture of human mammary epithelial stem cells. To summarize, this work reports for the first time that human mammary epithelial stem cells have an identical marker profile as breast cancer stem cells, which is CD24low/-CD44high. It has also been demonstrated for the first time that in long term mammosphere culture, the number of self renewal divisions of human mammary stem cells is restricted to four in vitro passages, at which most of the cells undergo senescence. Altering one of the parameters of the niche, by culturing mammospheres at physiological O2 level failed to prolong the in vitro lifespan of the spheres, although cell survival, proliferation and sphere formation increased, indicating that the niche requirements of human mammary epithelial stem cells for their long term self renewal needs to be further characterized.

NKX3.1, a prostate specific homeobox gene is a known marker of prostate epithelium during embryogenesis and is also expressed subsequently through different stages of prostate differentiation. However, all studies on NKX3.1 are focused on its regulation by androgen receptor (AR). The aim of this project is to establish the role of NKX3.1 in differentiation in prostate cancer, independent of AR regulation. In this thesis, I characterize the cell lines in terms of their differentiation capabilities in 3D, expression levels of NKX3.1 and the mismatch repair status. The genes potentially involved in differentiation and regulators of NKX3.1 are also identified using microarray data of the cell lines (<b>Chapter 3</b>). Although NKX3.1 plays a key role in prostate development no studies have been conducted on the effect of NKX3.1 expression on differentiation capabilities of prostate cell lines. In <b>Chapter 4</b>, this was investigated by siRNA mediated knockdown of NKX3.1 in 22Rv1 cell line and overexpression of NKX3.1 in PC3 (designated PC3-Nkx3.1) and PNT1a cells followed by growth in 3D. These functional studies show that the expression of NKX3.1 is vital for lumen formation in 3D, which is used as a measure of differentiation. The microarray data and overexpression of NKX3.1 studies suggest that this gene may also be involved in inhibiting epithelial to mesenchymal transition (EMT). Homeobox B13 (HOXB13) was identified as one of the downstream targets of NKX3.1. NKX3.1 and HOXB13 expression levels are positively correlated not only in the panel of prostate cell lines but also in the NKX3.1 overexpression and knockdown studies (<b>Chapter 5</b>). The results of the work presented in this thesis demonstrate that there is a striking parallel between the function of NKX3.1 in prostate and Caudal-type homeobox 1 (CDX1) in the colon and rectum. In conclusion, NKX3.1 plays a key role as a tumour suppressor in prostate cancer by controlling differentiation of prostate cancer cells.