Tetsuya Taga

The inhibitory effect of salinomycin on the proliferation, migration and invasion of human endometrial cancer stem-like cells

GOALS We previously demonstrated that side-population (SP) cells in human endometrial cancer cells (Hec1 cells) and in rat endometrial cells expressing oncogenic human K-Ras protein (RK12V cells) have features of cancer stem cells (CSCs). Hec1-SP cells showed enhanced migration and the potential to differentiate into the mesenchymal cell lineage. In this study, we analyzed the association of the epithelial-mesenchymal transition (EMT) with the properties of these endometrial CSCs. We also assessed the effects of salinomycin (a compound with EMT-specific toxicity) on the proliferative capacity, migration and invasiveness of these endometrial CSCs using Hec1-SP cells. METHOD We performed microarray expression analysis to screen for up-regulated genes in CSCs using a set of RK12V-SP cells and -non-SP(NSP) cells and used the Metacore package to identify the Gene GO pathway MAPs involved in the up-regulated genes. To analyze their association with EMT, the expression of several EMT associated genes in Hec1-SP cells was investigated by real time PCR and compared with that in Hec1-NSP cells. We assessed the expression of BAX, BCL2, LEF1, cyclinD and fibronectin by real time PCR. We also evaluated the viabilities, migration and invasive activities, and tumorigenicities of these SP cells and NSP cells in the presence or absence of salinomycin. RESULTS We demonstrated that i) EMT processes were observed in both RK12V-SP cells and Hec1-SP cells, ii) the level of fibronectin was enhanced in Hec1-SP cells and salinomycin reduced the level of fibronectin expression, iii) salinomycin induced apoptosis and inhibited Wnt signaling, and iv) salinomycin inhibited the proliferation, migration, invasiveness and tumorigenicity of these SP cells. CONCLUSION This is the first report of an inhibitory effect of salinomycin on the properties of endometrial CSCs.

Sox17 haploinsufficiency results in perinatal biliary atresia and hepatitis in C57BL/6 background mice

Congenital biliary atresia is an incurable disease of newborn infants, of unknown genetic causes, that results in congenital deformation of the gallbladder and biliary duct system. Here, we show that during mouse organogenesis, insufficient SOX17 expression in the gallbladder and bile duct epithelia results in congenital biliary atresia and subsequent acute ‘embryonic hepatitis’, leading to perinatal death in ~95% of the Sox17 heterozygote neonates in C57BL/6 (B6) background mice. During gallbladder and bile duct development, Sox17 was expressed at the distal edge of the gallbladder primordium. In the Sox17+/− B6 embryos, gallbladder epithelia were hypoplastic, and some were detached from the luminal wall, leading to bile duct stenosis or atresia. The shredding of the gallbladder epithelia is probably caused by cell-autonomous defects in proliferation and maintenance of the Sox17+/− gallbladder/bile duct epithelia. Our results suggest that Sox17 plays a dosage-dependent function in the morphogenesis and maturation of gallbladder and bile duct epithelia during the late-organogenic stages, highlighting a novel entry point to the understanding of the etiology and pathogenesis of human congenital biliary atresia.

Sox17-mediated maintenance of fetal intra-aortic hematopoietic cell clusters.

ABSTRACT During mouse development, definitive hematopoiesis is first detected around embryonic day 10.5 (E10.5) in the aorta-gonad-mesonephros (AGM) region, which exhibits intra-aortic cell clusters. These clusters are known to contain hematopoietic stem cells (HSCs). On the other hand, it is not clear how the cells in such clusters maintain their HSC phenotype and how they are triggered to differentiate. Here we found that an endodermal transcription factor marker, Sox17, and other F-group (SoxF) proteins, Sox7 and Sox18, were expressed in E10.5 intra-aortic cell clusters. Forced expression of any of these SoxF proteins, particularly Sox17, in E10.5 AGM CD45low c-Kithigh cells, which are the major component of intra-aortic clusters, led to consistent formation of cell clusters in vitro during several passages of cocultures with stromal cells. Cluster-forming cells with constitutive Sox17 expression retained long-term bone marrow reconstitution activity in vivo. Notably, shutdown of exogenously introduced Sox17 gene expression resulted in immediate hematopoietic differentiation. These results indicate that SoxF proteins, especially Sox17, contribute to the maintenance of cell clusters containing HSCs in the midgestation AGM region. Furthermore, SoxF proteins play a pivotal role in controlling the HSC fate decision between indefinite self-renewal and differentiation during fetal hematopoiesis.

A growth-promoting signaling component cyclin D1 in neural stem cells has antiastrogliogenic function to execute self-renewal

Self‐renewing proliferation of neural stem cells (NSCs) is intimately linked to the inhibition of neuronal and glial differentiation, however, their molecular linkage has been poorly understood. We have proposed a model previously explaining partly this linkage, in which fibroblast growth factor 2 (FGF2) and Wnt signals cooperate to promote NSC self‐renewal via β‐catenin accumulation, which leads to the promotion of proliferation by lymphoid enhancer factor (LEF)/T‐cell factor (TCF)‐mediated cyclin D1 expression and at the same time to the inhibition of neuronal differentiation by β‐catenin‐mediated potentiation of Notch signaling. To fully understand the mechanisms underlying NSC self‐renewal, it needs to be clarified how these growth factor signals inhibit glial differentiation as well. Here, we demonstrate that cyclin D1, a NSC growth promoting signaling component and also a common component of FGF2 and Wnt signaling pathways, inhibits astroglial differentiation of NSCs. Interestingly, this effect of cyclin D1 is mediated even though its cell cycle progression activity is blocked. Forced downregulation of cyclin D1 enhances astrogliogenesis of NSCs in culture and in vivo. We further demonstrate that cyclin D1 binds to STAT3, a transcription factor downstream of astrogliogenic cytokines, and suppresses its transcriptional activity on the glial fibrillary acidic protein (Gfap) gene. Taken together with our previous finding, we provide a novel molecular mechanism for NSC self‐renewal in which growth promoting signaling components activated by FGF2 and Wnts inhibit neuronal and glial differentiation. Stem Cells 2014;32:1602–1615

Glioma Stem Cells

Gliomas are the most frequent primary brain tumors and are classified as grade I to IV according to their degree of malignancy (Daumas-Duport et al., 1988). Grade I and II gliomas are clinically benign or semi-benign with relatively long-term survival, while grade III and IV are malignant and lethal within several years. In particular, glioblastoma multiforme (GBM), the most malignant glioma as grade IV often relapse even after radical surgical resection and standard chemo/radiation therapies, because of their diffuse infiltration into the surrounding brain parenchyma and high degree of chemo/radioresistance. Despite extensive efforts, the overall survival of GBM patients remains still short and has not yet been dramatically improved for more than several decades. GBMs are often composed of various types of cells with distinct morphology and clinical phenotypes. Their histological and biological multiformity has been classically explained by the stochastic clonal evolution model (Nowell, 1976). According to this model, all tumor cells should have low but inheritable ability to form tumors. However, recent evidence has suggested another concept, the cancer stem cell (CSC) hierarchy model, which shows that only a rare stem cell population has high ability to proliferate (Jordan et al., 2006). CSCs have similar characters to normal stem cells in the way of having high ability to self-renew and differentiate into multiple types of progenies to organize tissue architectures. Exclusively, CSCs can proliferate uncontrollably to propagate tumor cells. CSC model have direct relevance with tumor replenishment, disease recurrence and metastatic activity, suggesting that CSCs should be the target to eradicate the tumors. The aim of this chapter is to provide our insights into how CSCs in human glioma; i.e. glioma stem cells (GSCs) should be attacked. In the first parts, we summarize the general basis of CSC concept inclusive of the current knowledge on how a CSC is defined, how CSCs should be technically prepared and modeled, and what characteristics CSCs possess. In the following part, we highlight what abnormal signaling pathways regulate CSCs as the potential therapeutic targets. Understanding the framework of a GSC research field could help us to think of novel treatment strategy. Most importantly, however, CSCs have enhanced resistance to conventional chemotherapies. Considering this fact, we finally propose that it could be most promising strategy to disrupt the extracellular environments

Induction of protumoral CD11c(high) macrophages by glioma cancer stem cells through GM-CSF.

Cancer stem cells (CSCs) are maintained under special microenvironment called niche, and elucidation and targeting of the CSC niche will be a feasible strategy for cancer eradication. Tumor‐associated macrophages (TAMs) are known to be involved in cancer progression and thus can be a component of CSC niche. Although TAMs are known to play multiple roles in tumor progression, involvement of CSCs in TAM development fully remains to be elucidated. Using rat C6 glioma side population (SP) cells as a model of glioma CSCs, we here show that CSCs induce the TAM development by promoting survival and differentiation of bone marrow‐derived monocytes. CSC‐induced macrophages can be separated into two distinct subsets of cells, CD11clow and CD11chigh cells. Interestingly, only the CD11chigh subset of cells have protumoral activity, as shown by intracranial transplantation into immune‐deficient mice together with CSCs. These CD11chigh macrophages were observed in the tumor formed by co‐transplantation with CSCs. Furthermore, CSCs produced GM‐CSF and anti‐GM‐CSF antibody inhibited CSC‐induced TAM development. In conclusion, CSCs have the ability to self‐create their own niche involving TAMs through CSC‐derived GM‐CSF, which can thus be a therapeutic target in view of CSC niche disruption.

Identification and analysis of CXCR4-positive synovial sarcoma-initiating cells

Synovial sarcoma accounts for almost 10% of all soft tissue sarcomas, and its prognosis is poor with 5-year survival rates at 36%. Thus, new treatments and therapeutic targets for synovial sarcoma are required. Tumor-initiating cells have been defined by the ability for self-renewal and multipotent differentiation, and they exhibit higher tumorigenic capacity, chemoresistance and radiation resistance, expecting to be a new therapeutic target. In synovial sarcoma, the presence of such stemness remains largely unclear; thus, we analyzed whether synovial sarcoma possessed tumor-initiating cells and explored specific markers, and we discovered that synovial sarcoma cell lines possessed heterogeneity by way of containing a sphere-forming subpopulation highly expressing NANOG, OCT4 and SOX2. By expression microarray analysis, CXCR4 was identified to be highly expressed in the sphere subpopulation and correlated with stem-cell-associated markers. Inhibition of CXCR4 suppressed the cell proliferation of synovial sarcoma cell lines in vitro. The tumor-initiating ability of CXCR4-positive cells was demonstrated by xenograft propagation assay. CXCR4-positive cells showed higher tumorigenicity than negative ones and possessed both self-renewal and multipotent differentiation ability. Immunohistochemical analysis of 39 specimens of synovial sarcoma patients revealed that CXCR4 strongly correlated with poor prognosis of synovial sarcoma. Thus, we conclude that CXCR4 is the marker of synovial sarcoma-initiating cells, a new biomarker for prognosis and a new potential therapeutic target.

Enhancement of 5-aminolevulinic acid-based fluorescence detection of side population-defined glioma stem cells by iron chelation

Cancer stem cells (CSCs) are dominantly responsible for tumor progression and chemo/radio-resistance, resulting in tumor recurrence. 5-aminolevulinic acid (ALA) is metabolized to fluorescent protoporphyrin IX (PpIX) specifically in tumor cells, and therefore clinically used as a reagent for photodynamic diagnosis (PDD) and therapy (PDT) of cancers including gliomas. However, it remains to be clarified whether this method could be effective for CSC detection. Here, using flow cytometry-based analysis, we show that side population (SP)-defined C6 glioma CSCs (GSCs) displayed much less 5-ALA-derived PpIX fluorescence than non-GSCs. Among the C6 GSCs, cells with ultralow PpIX fluorescence exhibited dramatically higher tumorigenicity when transplanted into the immune-deficient mouse brain. We further demonstrated that the low PpIX accumulation in the C6 GSCs was enhanced by deferoxamine (DFO)-mediated iron chelation, not by reserpine-mediated inhibition of PpIX-effluxing ABCG2. Finally, we found that the expression level of the gene for heme oxygenase-1 (HO-1), a heme degradation enzyme, was high in C6 GSCs, which was further up-regulated when treated with 5-ALA. Our results provide important new insights into 5-ALA-based PDD of gliomas, particularly photodetection of SP-defined GSCs by iron chelation based on their ALA-PpIX-Heme metabolism.

Requirement of ABC transporter inhibition and Hoechst 33342 dye deprivation for the assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes

BackgroundElucidating the precise properties of cancer stem cells (CSCs) is indispensable for the development of effective therapies against tumors, because CSCs are key drivers of tumor development, metastasis and relapse. We previously reported that the Hoechst 33342 dye-low staining side population (SP) method can enrich for CSCs in the C6 glioma cell line, and that the positively stained main population (MP) cells are non-CSCs. Presence of cancer stem-like SP cells is reported in various types of cancer. Although altered cellular energy metabolism is a hallmark of cancer, very little has been studied on the applicability of fluorescent probes for the understanding of CSC energy metabolism.MethodsThe metabolic status of C6 SP and MP cells are evaluated by CellROX, MitoTracker Green (MTG) and JC-1 for cellular oxidative stress, mitochondrial amount, and mitochondrial membrane potential, respectively.ResultsSP cells were found to exhibit significantly lower fluorescent intensities of CellROX and MTG than MP cells. However, inhibition of ATP binding cassette (ABC) transporters by verapamil enhanced the intensities of these probes in SP cells to the levels similar to those in MP cells, indicating that SP cells expel the probes outside of the cells through ABC transporters. Next, SP cells were stained with JC-1 dye which exhibits membrane potential dependent accumulation in mitochondrial matrix, followed by formation of aggregates. The mitochondrial membrane potential indicated by the aggregates of JC-1 was 5.0-fold lower in SP cells than MP cells. Inhibition of ABC transporters enhanced the fluorescent intensities of the JC-1 aggregates in both SP and MP cells, the former of which was still 2.2-fold lower than the latter. This higher JC-1 signal in MP cells was further found to be due to the Hoechst 33342 dye existing in MP cells. When SP and MP cells were recultured to deprive the intracellular Hoechst 33342 dye and then stained with JC-1 in the presence of verapamil, the intensities of JC-1 aggregates in such SP and MP cells became comparable.ConclusionInhibiting ABC transporters and depriving Hoechst 33342 dye are required for the accurate assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

Sox17 as a candidate regulator of myeloid restricted differentiation potential

Sry related high mobility group box 17 (Sox17), which is a marker of endodermal cells and a transcriptional regulator, has a critical role in the maintenance of fetal and neonatal hematopoietic stem cells (HSC). Sox17 has been identified as a key regulator of the development and differentiation of fetal hematopoietic progenitors from the aorta‐gonad‐mesonephros (AGM) region. The co‐culture of Sox17‐transduced hematopoietic progenitor cells (CD45lowc‐Kithigh cells) from AGM regions on OP9 stromal cells gives rise to multipotential hematopoietic stem/progenitor cells. Here, we show that in a primary transplantation experiment, Sox17‐transduction in CD45lowc‐Kithigh cells of embryonic day (E) 10.5 AGM increased the absolute number of common myeloid progenitors (CMPs) in the bone marrow (BM) of recipient mice in comparison to that of granulocyte/macrophage progenitors (GMPs) and the megakaryocyte/erythroid progenitors (MEPs). When Sox17‐transduced cells were cultured with OP9 stromal cells, Sox17‐transduced GMPs (Sox17‐GMPs), Sox17‐transduced CMPs (Sox17‐CMPs), and Sox17‐transduced MEPs (Sox17‐MEPs) were generated. Sox17‐GMPs and Sox17‐CMPs maintained their self‐renewal capacity and the hematopoietic ability upon co‐culture with the OP9 stromal cells for some passages. Moreover, Sox17‐GMPs exhibited the increase in expression of c‐Mpl and GATA‐2 in comparison to GMPs of BM and Sox17‐CMPs showed the increase in expression of c‐Mpl, NF‐E2, and β‐globin genes in comparison to CMPs of BM. Furthermore, when Sox17‐transduced cells were cultured in methylcellulose to examine the colony‐forming ability, Sox17‐GMPs and Sox17‐CMPs maintained the formation of mixed colonies for some passages. Taken together, Sox17 is suggested to regulate the maintenance and differentiation of hematopoietic progenitors derived from AGM regions at midgestation, in particular myeloid progenitors.