Instructive signals, whether external or internal, play critical roles in regulating the developmental potency or ability to self-renew of stem cells. External signals may range from secreted growth factors to extracellular matrix proteins found in the stem cell niche. Internal signals include activated signalling cascades and the eventual transcriptional mechanisms they initiate. In either fashion, stem cells are regulated in a complex temporal and context specific manner in order to maintain or maximise their unique characteristics. Previous experiments suggest that Wnt3a plays a role in maintaining the pluripotent state of mouse embryonic stem (mES) cells. However, in the absence of leukemia inhibitory factor (LIF), Wnt signalling is unable to maintain ES cells in the undifferentiated state. This implies that maintaining the pluripotent state of mES cells is not the primary function of canonical Wnt signalling. To further characterize the role of Wnt3a in pluripotency and lineage specification undifferentiated and differentiated mES cells were induced with Wnt3a. Wnt3a induced the formation of a metastable primitive endoderm state and upon subsequent differentiation, the induction of large quantities of visceral endoderm. Furthermore, we determined that the ability of Wnt3a to induce a metastable primitive endoderm state was mediated by the T-box transcription factor Tbx3. Our data demonstrates a novel role for Wnt3a in promoting the interconversion of undifferentiated mES cells into a pluripotent primitive endoderm state. Aging of skeletal muscle tissue is accompanied by fibrosis, atrophy and remodeling all of which negatively affect muscle performance. Whether this reduction in skeletal muscle competency is directly attributed to a resident adult stem cell population called satellite cells remains largely unknown. Here, we undertook an investigation into how age affects the transcriptional profile of satellite cells and their repopulating ability following transplantation. We determined that as satellite cells age, both their regenerative capacity and ability to colonize the satellite cell niche is reduced. Additionally, we identified satellite cell specific transcriptional profiles that differed with respect to age. Therefore, we conclude that intrinsic factors are an important determinant of satellite cell regenerative capacity during the aging process.

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.

Medulloblastoma (MB), a cancer of the cerebellum, is the most common solid tumor affecting children. In the cerebellum, Sonic Hedgehog (Shh) drives the proliferative expansion of granule neuron progenitors (GNP). These cells are located in the external granule layer (EGL) and are the cells of origin of Shh-MB. We recently identified Norrie Disease Pseudoglioma (Ndp) as a novel downstream target of Hh signaling in the developing retina. Ndp encodes an X-linked cysteine-rich secreted protein called Norrin, which is best known for its role in angiogenesis and blood brain barrier (BBB) maintenance in the developing retina and cerebellum, respectively. Norrin mediates this effect by binding to its receptor Frizzled4 (Fzd4) and co-receptors LRP5/6 and Tpsan12 to activate the canonical, β-catenin-dependent Wnt signaling pathway in endothelial cells (ECs). We detected the expression of Ndp and all required receptors in mouse GNPs and MB samples. To investigate a potential role for Ndp in Hh-driven MB, we genetically and pharmacologically inactivated Ndp/Fzd4 signaling in Ptch+/- mice (a mouse model for human Gorlin syndrome), which dramatically increased the incidence and reduced the latency of MB. This accelerated rate of tumorigenesis was caused by an increase in the number of preneoplastic lesions (PNLs), the precursor lesions to MB, and a faster conversion of these lesions to MB. We showed that Ndp mediates this increase in tumorigenesis by signaling through endothelial cell receptor Fzd4 to alter the GNP stroma, which is characterised by 5 major alterations: 1) activated angiogenic program, 2) open BBB, 3) aberrant deposition of extracellular matrix, 4) aberrant lymphocyte recruitment and 5) reduction in meningeal lymphatic vasculature. We propose that these stromal alterations are associated with a pro-tumor microenvironment that promotes DNA damage in GNPs and leads to enhanced lesion formation and progression towards MB. This research highlights 1) an unanticipated role for Ndp/Fzd4 signaling in Shh-MB initiation and progression, 2) a role for stromal signaling in the regulation of MB development and 3) a previously undescribed role for Ndp signaling in maintaining meningeal cerebellum lymphatic vessels.

Wnt/β-catenin signaling is activated in the heart after myocardial infarction (MI). This study aims to investigate if β-catenin deletion affects post-MI ion channel gene alterations and ventricular tachycardias (VT). MI was induced by permanent ligation of left anterior descending artery in wild-type (WT) and cardiomyocyte-specific β-catenin knockout (KO) mice. KO mice showed reduced susceptibility to VT (18% vs. 77% in WT) at 8 weeks after MI, associated with reduced scar size and attenuated chamber dilation. qPCR analyses of both myocardial tissues and purified cardiomyocytes demonstrated upregulation of Wnt pathway genes in border and infarct regions after MI, including Wnt ligands (such as Wnt4) and receptors (such as Fzd1 and Fzd2). At 1 week after MI, cardiac sodium channel gene (Scn5a) transcript was reduced in WT but not in KO hearts, consistent with previous studies showing Scn5a inhibition by Wnt/β-catenin signaling. At 8 weeks after MI when Wnt genes have declined, Scn5a returned to near sham levels and K⁺ channel gene downregulations were not different between WT and KO mice. This study demonstrated that VT susceptibility in the chronic phase after MI is reduced in mice with cardiomyocyte-specific β-catenin deletion primarily through attenuated structural remodeling, but not ion channel gene alterations.

Background: The canonical Wnt/β-catenin pathway is activated in a variety of heart diseases, such as myocardial infarction and cardiac hypertrophy, that are associated with altered ion channel expressions and increased risk of cardiac arrhythmias. Previous work from our lab has demonstrated that the Wnt/β-catenin signalling (Wnt signalling) inhibits sodium (Na+) current in rat cardiomyocytes. In this project, we aim to investigate the mechanisms that underlie the inhibition of Na+ current by Wnt signalling in both rat and human cardiomyocytes. Results: In both neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), activation of the Wnt/β-catenin signalling led to reduced level of Na+ channel gene transcript (Scn5a), channel protein (Nav1.5) and channel current density. This suggests that reduced Scn5a expression is likely the primary mechanism for reduced Na+ current. In addition, we found that activation of the Wnt/β-catenin signalling in both NRVMs and iPSC-CMs upregulated Tbx3 transcript and protein levels, which is a transcription factor that is known to suppress Scn5a transcription. In NRVMs, siRNA-mediated Tbx3 knockdown attenuated (by ~30%) Wnt-induced reductions in Scn5a and Nav1.5 levels. Conclusions: Our findings are consistent with the conclusion that Wnt/β-catenin signalling inhibits Na+ current in both rat and human cardiomyocytes by reducing Scn5a levels, with Tbx3 as one of the mediators.

Gastrin and its precursors promote proliferation in different gastrointestinal cells. Since mature, amidated gastrin (G-17) can induce cyclin D1, we determined whether G-17-mediated induction of cyclin D1 transcription involved Wnt signaling and CRE-binding protein (CREB) pathways. Our studies indicate that G-17 induces protein, mRNA expression and transcription of the G1-specific marker cyclin D1, in the gastric adenocarcinoma cell line AGSE (expressing the gastrin/cholecystokinin B receptor). This was associated with an increase in steady-state levels of total and nonphospho β-catenin and its nuclear translocation, indicating the activation of the Wnt-signaling pathway. In addition, G-17-mediated increase in cyclin D1 transcription was significantly attenuated by axin or dominant-negative (dn) T-cell factor 4(TCF4), suggesting crosstalk of G-17 with the Wnt-signaling pathway. Mutational analysis indicated that this effect was mediated through the cyclic AMP response element (CRE) (predominantly) and the TCF sites in the cyclin D1 promoter, which was also inhibited by dnCREB. Furthermore, G-17 stimulation resulted in increased CRE-responsive reporter activity and CREB phosphorylation, indicating an activation of CREB. Chromatin immunoprecipitation studies revealed a G-17-mediated increase in the interaction of β-catenin with cyclin D1 CRE, which was attenuated by dnTCF4 and dnCREB. These results indicate that G-17 induces cyclin D1 transcription, via the activation of β-catenin and CREB pathways.

Indiana University-Purdue University Indianapolis (IUPUI) / Intervertebral discs (IVD) degenerate earlier than many other musculoskeletal tissues and will continue to degenerate with aging. IVD degeneration affects up to 80 percent of the adult population and is a major contributing factor to low back pain. Anti-sclerostin antibody is an FDA-approved treatment for osteoporosis in postmenopausal women at high-risk for fracture and, as a systemic stimulant of the Wnt/LRP5/β-Catenin signaling pathway, may impact the IVD. Stabilization of β-Catenin in the IVD increases Wnt signaling and is anabolic to the extracellular matrix (ECM), while deletion of β-catenin or LRP5 decreases Wnt signaling and is catabolic to the ECM. Here, we hypothesized that a reduction of Sost would stimulate ECM anabolism. Lumbar and caudal (tail) IVD and vertebrae of Sost KO and WT (wildtype) mice (n=8 each) were harvested at 16 weeks of age and tested by MRI, histology, immunohistochemistry, Western Blot, qPCR, and microCT. Compared to WT, Sost KO reduced sclerostin protein and Sost gene expression. Next, Sost KO increased the hydration of the IVD and the proteoglycan stain in the nucleus pulposus and decreased the expression of genes associated with IVD degeneration, e.g., heat shock proteins. However, deletion of Sost was compensated by less unphosphorylated (active) β-Catenin protein in the cell nucleus, upregulation of Wnt signaling inhibitors Dkk1 and sFRP4, and catabolic ECM gene expression. Consequently, notochordal and early chondrocyte-like cells (CLCs) were replaced by mature CLCs. Overall, Sost deletion increased hydration and proteoglycan protein content, but activated a compensatory suppression of Wnt signaling that may trigger chondrogenesis and may potentially be iatrogenic to the IVD in the long-term.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18149号 / 医博第3869号 / 新制||医||1002(附属図書館) / 31007 / 京都大学大学院医学研究科医学専攻 / (主査)教授 福田 和彦, 教授 渡邉 大, 教授 河野 憲二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Metastasis suppressors regulate multiple steps during the process of dissemination of tumor cells from primary sites to distant organs, while they do not affect the growth of the primary tumor. Previously, we identified NDRG1 (N-myc downstream regulated gene 1) as a tumor metastasis suppressor gene and found that it is negatively involved in metastatic progression of prostate and breast cancers. To elucidate the molecular mechanism of NDRG1 function, we used the yeast two-hybrid system to identify proteins interacting with NDRG1. In the first part of this project, we demonstrate that NDRG1, interacts with the Wnt receptor, LRP6, followed by blocking of the Wnt signaling, and therefore, orchestrates a cellular network that impairs the metastatic progression of tumor cells in vitro and in animal model. We also found that restoring NDRG1 expression by a small molecule compound significantly suppressed the capability of otherwise highly metastatic tumor cells to thrive in circulation and distant organs in animal models. In addition, our analysis of clinical cohorts data indicate that Wnt+/NDRG-/LRP+ signature has a strong predictable value for recurrence-free survival of cancer patients. Collectively, we have identified NDRG1 as a negative master regulator of Wnt signaling during the metastatic progression, and therefore revealed a novel control mechanism of Wnt signaling in tumor progression. Previously, we identified the metastasis promoting transcription factor, ATF3, as a downstream target of NDRG1. Further analysis revealed that the KAI1 promoter contained a consensus binding motif of ATF3, suggesting a possibility that NDRG1 suppresses metastasis through inhibition of ATF3 expression followed by activation of KAI1 gene. In the second part of this project, we examine a possible link between two metastasis suppressor genes, NDRG1 and KAI1, through ATF3. We demonstrated that ectopic expression of NDRG1 was able to augment endogenous KAI1gene expression in prostate cancer cell lines, while silencing NDRG1 accompanied with significant decrease in KAI1 expression in vitro and in vivo. In addition, our results of ChIP analysis indicate that ATF3 indeed bound to the promoter of KAI1 gene. Importantly, our promoter-based analysis revealed that ATF3 modulated KAI1 transcription through cooperation with other endogenous transcription factor as co-activator (ATF3-JunB) or co-repressor (ATF3-NFêB). Moreover, loss of KAI1 expression significantly abrogated NDRG1-mediated metastatic suppression in vitro as well as in a spontaneous metastasis animal model, indicating that KA11 is a functional down-stream target of NDRG1 pathway. Our result of immunohistochemical analysis showed that loss of NDRG1 and KAI1 occurs in parallel as prostate cancer progresses. We also found that a combined expression status of these two genes serves as a strong independent prognostic marker to predict metastasis-free survival of prostate cancer patients. Taken together, our result revealed a novel regulatory network of two metastasis suppressor genes, NDRG1 and KAI1, which together concerted metastasis-suppressive activities through intrinsic transcriptional cascade.

SRY (sex determining region Y)-box 2 (SOX2) is one the embryonic stem cell transcription factors that is capable of reprogramming adult differentiated cells into an induced pluripotent cell. SOX2 is amplified in various types of epithelial cancers and its high its expression correlates with poor prognosis and decreased patient survival. Aberrant Wnt signaling drives the colo-rectal carcinogenic process and is a major determinant of the disease outcome. This study demonstrates that SOX2 counteracts Wnt driven tumor cell proliferation and maintains quiescence in a sub-population of Colo-Rectal Cancer (CRC) cells. High SOX2 expression is found in a sub-group of CRC patients with advanced disease. High SOX2 expression coupled with low Wnt activity was found in SW620 metastatic CRC cell line, while the opposite was true for the isogenic SW480 primary tumor cell line. SOX2 silencing increased Wnt activity and enhanced the oncogenic potential of SW620 cells in vitro and in vivo while over-expression had opposite effects in SW480 cells. SOX2 up-regulates the expression of PTPRK and PHLPP2 protein phosphatase genes which in turn attenuates Wnt activity by interfering with Protein Kinase A, B and C mediated beta catenin phosphorylation at Serine 552 and 675 amino acid residues thereby diminishing its nuclear sequestration and transcriptional activation. Thus SOX2 mitigates growth factor mediated Wnt activation in CRC cells and inhibits cellular proliferation so that these cells are forced to change their oncogene addiction. In effect, high SOX2 expression causes clonal evolution of APC mutant CRC cells from a state of high Wnt dependency to a state of low Wnt dependency in the process making such cells resistant to Wnt inhibitor therapy. Enhanced SOX2 transcriptional activity was associated with increased proportion of cancer cells in G0-G1 phase of cell cycle. Changing SOX2 protein levels in cells had a direct correlation with mRNA levels of RBL2-HUMAN and CDKN2B genes, which serve as regulators of G0 and G1 respectively. SOX2 was shown to physically bind and to the promoter region of these two genes and enhance their transcription. Thus high SOX2 expression, up-regulates the expression of key cell cycle inhibitor genes like RBL2 and CDKN2B and keeps cells in a dormant state. This phenomenon allows colon cancer cells to escape from cytotoxic drug therapy directed at rapidly dividing cells and cause treatment failure and disease relapse.