Establishment and characterization of a novel cancer stem-like cell of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive malignant tumor of bile duct epithelia. Recent evidence suggests the impact of cancer stem cells (CSC) on the therapeutic resistance of CCA; however, the knowledge of CSC in CCA is limited due to the lack of a CSC model. In this study, we successfully established a stable sphere-forming CCA stem-like cell, KKU-055-CSC, from the original CCA cell line, KKU-055. The KKU-055-CSC exhibits CSC characteristics, including: (1) the ability to grow stably and withstand continuous passage for a long period of culture in the stem cell medium, (2) high expression of stem cell markers, (3) low responsiveness to standard chemotherapy drugs, (4) multilineage differentiation, and (5) faster and constant expansive tumor formation in xenograft mouse models. To identify the CCA-CSC-associated pathway, we have undertaken a global proteomics and functional cluster/network analysis. Proteomics identified the 5925 proteins in total, and the significantly upregulated proteins in CSC compared with FCS-induced differentiated CSC and its parental cells were extracted. Network analysis revealed that high mobility group A1 (HMGA1) and Aurora A signaling through the signal transducer and activator of transcription 3 pathways were enriched in KKU-055-CSC. Knockdown of HMGA1 in KKU-055-CSC suppressed the expression of stem cell markers, induced the differentiation followed by cell proliferation, and enhanced sensitivity to chemotherapy drugs including Aurora A inhibitors. In silico analysis indicated that the expression of HMGA1 was correlated with Aurora A expressions and poor survival of CCA patients. In conclusion, we have established a unique CCA stem-like cell model and identified the HMGA1-Aurora A signaling as an important pathway for CSC-CCA.

Targeting alpha2,3-sialylated glycan in glioma stem-like cells by Maackia amurensis lectin-II: A promising strategy for glioma treatment.

Glioma stem/initiating cells have been considered a major cause of tumor recurrence and therapeutic resistance. In this study, we have established a new glioma stem-like cell (GSC), named U373-GSC, from the U373 glioma cell line. The cells exhibited stemness properties, e.g., expression of stem cell markers, self-renewal activity, multi-lineage differentiating abilities, and drug resistance. Using U373-GSC and GSC-03A-a GSC clone previously established from patient tissue, we have identified a novel GSC-associated sialic acid-modified glycan commonly expressed in both cell lines. Lectin fluorescence staining showed that Maackia amurensis lectin II (MAL-II)-binding alpha2,3-sialylated glycan (MAL-SG) was highly expressed in GSCs, and drastically decreased during FBS induced differentiation to glioma cells or little in the parental cells. Treatment of GSCs by MAL-II, compared with other lectins, showed that MAL-II significantly suppresses cell viability and sphere formation via induction of cell cycle arrest and apoptosis of the GSCs. Similar effects were observed when the cells were treated with a sialyltransferase inhibitor or sialidase. Taken together, we demonstrate for the first time that MAL-SGs/alpha-2,3 sialylations are upregulated and control survival/maintenances of GSCs, and their functional inhibitions lead to apoptosis of GSCs. MAL-SG could be a potential marker and therapeutic target of GSCs; its inhibitors, such as MAL-II, may be useful for glioma treatment in the future.

Ribosome Incorporation Induces EMT-like Phenomenon with Cell Cycle Arrest in Human Breast Cancer Cell.

Although ribosomes are generally known to be a translational machinery, some ribosomal proteins also have accessory functions involving early development and differentiation. Previously, we reported that ribosome incorporation into human dermal fibroblasts generated embryoid body-like cell clusters, altered cellular fate, and differentiated into cells of all 3 germ layers. However, the molecular phenomena induced by ribosome incorporation in the cell remained unknown. Here, we demonstrate that ribosome incorporation into human breast cancer cell MCF7 leads to ribosome-induced cell clusters (RICs) formation accompanying with epithelial-mesenchymal transition (EMT)-like gene expression. Following ribosome incorporation, MCF7 cells cease proliferation, which is caused by inhibition of cell cycle transition from G0 to G1 phase. Further, MCF7 RICs show induced expression of EMT markers, TGF-ß1 and Snail along with autophagy markers and tumor suppressor gene p53. These findings indicate that the incorporation of ribosome into cancer cells induces an EMT-like phenomenon and changes the cancer cell characteristics.

Ribosomal protein S6 promotes stem-like characters in glioma cells.

Glioblastoma multiforme (GBM), a lethal brain tumor developing in the white matter of the adult brain, contains a small population of GBM stem cells (GSCs), which potentially cause chemotherapeutic resistance and tumor recurrence. However, the mechanisms underlying the pathogenesis and maintenance of GSCs remain largely unknown. A recent study reported that incorporation of ribosomes and ribosomal proteins into somatic cells promoted lineage trans-differentiation toward multipotency. This study aimed to investigate the mechanism underlying stemness acquisition in GBM cells by focusing on 40S ribosomal protein S6 (RPS6). RPS6 was significantly upregulated in high-grade glioma and localized at perivascular, perinecrotic, and border niches in GBM tissues. siRNA-mediated RPS6 knock-down significantly suppressed the characteristics of GSCs, including their tumorsphere potential and GSC marker expression; STAT3 was downregulated in GBM cells. RPS6 overexpression enhanced the tumorsphere potential of GSCs and these effects were attenuated by STAT3 inhibitor (AG490). Moreover, RPS6 expression was significantly correlated with SOX2 expression in different glioma grades. Immunohistochemistry data herein indicated that RPS6 was predominant in GSC niches, concurrent with the data from IVY GAP databases. Furthermore, RPS6 and other ribosomal proteins were upregulated in GSC-predominant areas in this database. The present results indicate that, in GSC niches, ribosomal proteins play crucial roles in the development and maintenance of GSCs and are clinically associated with chemoradioresistance and GBM recurrence.

Tsukushi is essential for proper maintenance and terminal differentiation of mouse hippocampal neural stem cells.

Secreted proteoglycan molecule Tsukushi (TSK) regulates various developmental processes, such as early body patterning and neural plate formation by interacting with major signaling pathways like Wnt, BMP, Notch etc. In central nervous system, TSK inhibits Wnt signaling to control chick retinal development. It also plays important roles for axon guidance and anterior commissure formation in mouse brain. In the present study, we investigated the role of TSK for the development and proper functioning of mouse hippocampus. We found that TSK expression is prominent at hippocampal regions of early postnatal mouse until postnatal day 15 and gradually declines at later stages. Hippocampal dimensions are affected in TSK knockout mice (TSK-KO) as shown by reduced size of hippocampus and dentate gyrus (DG). Interestingly, neural stem cell (NSC) density at the neural niche of DG was higher in TSK-KO compared with wild-type. The ratio of proliferating NSCs as well as the rate of overall cell proliferation was also higher in TSK-KO hippocampus. Our in vitro study also suggests an increased number of neural stem/progenitor cells residing in TSK-KO hippocampus. Finally, we found that the terminal differentiation of NSCs in TSK-KO was disturbed as the differentiation to neuronal cell lineage was increased while the percentages of astrocytes and oligodendrocytes were decreased. Overall, our study establishes the involvement of TSK in hippocampal development, NSC maintenance and terminal differentiation at perinatal stages.

Akhirin regulates the proliferation and differentiation of neural stem cells/progenitor cells at neurogenic niches in mouse brain.

Specialized microenvironment, or neurogenic niche, in embryonic and postnatal mouse brain plays critical roles during neurogenesis throughout adulthood. The subventricular zone (SVZ) and the dentate gyrus (DG) of hippocampus in the mouse brain are two major neurogenic niches where neurogenesis is directed by numerous regulatory factors. Now, we report Akhirin (AKH), a stem cell maintenance factor in mouse spinal cord, plays a pivotal regulatory role in the SVZ and in the DG. AKH showed specific distribution during development in embryonic and postnatal neurogenic niches. Loss of AKH led to abnormal development of the ventricular zone and the DG along with reduction of cellular proliferation in both regions. In AKH knockout mice (AKH-/- ), quiescent neural stem cells (NSCs) increased, while proliferative NSCs or neural progenitor cells decreased at both neurogenic niches. In vitro NSC culture assay showed increased number of neurospheres and reduced neurogenesis in AKH-/- . These results indicate that AKH, at the neurogenic niche, exerts dynamic regulatory role on NSC self-renewal, proliferation and differentiation during SVZ and hippocampal neurogenesis.

Sirtuin 7 is involved in the consolidation of fear memory in mice.

Sirtuin 7 (SIRT7) is an NAD+-dependent deacetylase/deacylase, and is involved in a variety of biological processes relevant to the transcription of rRNA, the DNA damage response, tumorigenesis, and metabolism. SIRT7 mRNA is expressed ubiquitously, including in the brain, but there is no detailed information about the anatomical distribution and functional role of SIRT7 in the brain. Here, we demonstrated that SIRT7 is widely expressed in the mouse brain, including in the cortex, striatum, thalamus, hippocampus, and amygdala. Behavioral examinations revealed that Sirt7 knockout (KO) and control mice showed similar levels of freezing behavior immediately after a fear response, but a significant decrease of freezing behavior at 24 h after fear conditioning was observed in Sirt7 KO mice. Histological analysis revealed that there is no apparent structural abnormality of the amygdala and hippocampus, which are regions involved in fear memory consolidation, in Sirt7 KO mice. Our results indicate that SIRT7 is involved in the consolidation of fear memory.

Transdifferentiation of human somatic cells by ribosome.

Ribosomes are intracellular organelles ubiquitous in all organisms, which translate information from mRNAs to synthesize proteins. They are complex macromolecules composed of dozens of proteins and ribosomal RNAs. Other than translation, some ribosomal proteins also have side-jobs called "Moonlighting" function. The majority of these moonlighting functions influence cancer progression, early development and differentiation. Recently, we discovered that ribosome is involved in the regulation of cellular transdifferentiation of human dermal fibroblasts (HDFs). In vitro incorporation of ribosomes into HDFs arrests cell proliferation and induces the formation of cell clusters, that differentiate into three germ layer derived cells upon induction by differentiation mediums. The discovery of ribosome induced transdifferentiation, that is not based on genetic modification, find new possibilities for the treatment of cancer and congenital diseases, as well as to understand early development and cellular lineage differentiation.

Regulation of the neural niche by the soluble molecule Akhirin.

Though the adult central nervous system has been considered a comparatively static tissue with little turnover, it is well established today that new neural cells are generated throughout life. Neural stem/progenitor cells (NS/PCs) can self-renew and generate all types of neural cells. The proliferation of NS/PCs, and differentiation and fate determination of PCs are regulated by extrinsic factors such as growth factors, neurotrophins, and morphogens. Although several extrinsic factors that influence neurogenesis have already been reported, little is known about the role of soluble molecules in neural niche regulation. In this review, we will introduce the soluble molecule Akhirin and discuss its role in the eye and spinal cord during development.

Tsukushi expression is dependent on Notch signaling and oscillated in the presomitic mesoderm during chick somitogenesis.

During somitogenesis, segmentation of the body axis occurs by epithelial somites budding off from the rostral end of the unsegmented presomitic mesoderm (PSM), and its molecular regulation is achieved by a molecular oscillator and signaling molecules. Tsukushi (TSK) is a unique secreted protein and involved in diverse biological cascades in vertebrate embryos by modulating several signaling pathways at the extracellular region. However, the involvement of TSK in somitogenesis remains unknown. In this study, we investigated the detailed expression patterns of TSK at different developmental stages of a chick embryo. Chick-TSK (C-TSK) is expressed in the PSM and shows an oscillation pattern with three phases. The oscillation pattern of C-TSK in the PSM is similar to that of c-Notch1 and c-hairy1, but not to c-Delta1. Our in vitro data showed that Notch signaling is necessary for the normal expression of C-TSK and that expression of C-TSK is an intrinsic property of the anterior PSM. These data suggest that TSK plays a role in chick somitogenesis.

Reprogramming of human somatic cells by bacteria.

In general, it had been believed that the cell fate restriction of terminally differentiated somatic cells was irreversible. In 1952, somatic cell nuclear transfer (SCNT) was introduced to study early embryonic development in frogs. So far, various mammalian species have been successfully cloned using the SCNT technique, though its efficiency is very low. Embryonic stem (ES) cells were the first pluripotent cells to be isolated from an embryo and have a powerful potential to differentiate into more than 260 types of cells. The generation of induced pluripotent stem (iPS) cells was a breakthrough in stem cell research, and the use of these iPS cells has solved problems such as low efficiency and cell fate restriction. These cells have since been used for clinical application, disease investigation, and drug selection. As it is widely accepted that the endosymbiosis of Archaea into eukaryotic ancestors resulted in the generation of eukaryotic cells, we examined whether bacterial infection could alter host cell fate. We previously showed that when human dermal fibroblast (HDF) cells were incorporated with lactic acid bacteria (LAB), the LAB-incorporated HDF cells formed clusters and expressed a subset of common pluripotent markers. Moreover, LAB-incorporated cell clusters could differentiate into cells derived from each of the three germinal layers both in vivo and in vitro, indicating successful reprogramming of host HDF cells by LAB. In the current review, we introduce the existing examples of cellular reprogramming by bacteria and discuss their nuclear reprogramming mechanisms.

The combinatorial guidance activities of draxin and Tsukushi are essential for forebrain commissure formation.

We have shown that draxin is a repulsive axon guidance molecule for a variety of neuron classes and that genetic deletion of draxin in mice results in the absence of all forebrain commissures. Moreover, we also identified a secreted molecule, Tsukushi (TSK), that belongs to the small leucine-rich proteoglycan family (SLRP) and inhibits signaling molecules, such as BMP and Wnt. TSK knockout mice show malformation of the corpus callosum (CC) and agenesis of the anterior commissure (AC), suggesting the importance of TSK function in forebrain commissure formation. There is a possibility that the combined function of these two proteins is essential for the formation of these commissures. In this study, we investigate this possibility by generating draxin/TSK doubly heterozygous mice and comparing their forebrain commissure phenotypes with those of singly heterozygous mice. We found that, although draxin and TSK did not interact directly, their genetic interaction was evident from the significantly higher prevalence of CC malformation and agenesis of the AC in the draxin/TSK doubly heterozygous mice. Importantly, in this study, we demonstrated a new function of TSK in guiding anterior olfactory neuronal (AON) and cortical axons. TSK bound to and provided growth inhibitory signals dose-dependently to AON and cortical axons in outgrowth assay. TSK also induced growth cone collapse when applied acutely to these cultured neurons. Furthermore, TSK and draxin had additive effects in inhibiting cortical and AON neurite outgrowth. Thus, based on a combination of genetic analyses and in vitro experiments, we propose that the combined guidance activities of draxin and TSK regulate forebrain commissure formation.

Inhibitory effects of draxin on axonal outgrowth and migration of precerebellar neurons.

The rhombic lip, a dorsal stripe of the neuroepithelium lining the edge of the fourth ventricle, is the site of origin of precerebellar neurons (PCN), which migrate tangentially towards the floor plate. After reaching the floor plate, they project their axons to the cerebellum. Although previous studies have shown that the guidance molecules Netrin/DCC and Slit/Robo have critical roles in PCN migration, the molecular mechanisms underlying this process remain poorly understood. Here, we report that draxin, a repulsive axon guidance protein, is involved in PCN development. We found that draxin is expressed in the rhombic lip and migratory stream of some PCN in the developing hindbrain of mice. In addition, draxin inhibited neurite outgrowth and nuclei migration from rhombic lip explants. These results suggest that draxin functions as a repulsive guidance cue for PCN migration. However, we observed no significant differences in PCN distribution between draxin(-/-) and wild type embryos. Thus, draxin and other axon guidance cues may have redundant roles in PCN migration.

Multiple roles of Equarin during lens development.

Since the days of Hans Spemann, the ocular lens has served as one of the most important developmental systems for elucidating the fundamental processes of induction and differentiation. Lens is an important source of signals that influence the eye development and a variety of genes expressed by the lens have been identified. The identification of additional molecule(s), especially secreted ones that might mediate signals, will extend our knowledge of the molecular mechanisms of eye and lens development. Here, we will introduce a soluble molecule, Equarin, and discuss its vital role in multiple aspects of lens development.

Tsukushi functions as a Wnt signaling inhibitor by competing with Wnt2b for binding to transmembrane protein Frizzled4.

The Wnt signaling pathway is essential for the development of diverse tissues during embryogenesis. Signal transduction is activated by the binding of Wnt proteins to the type I receptor low-density lipoprotein receptor-related protein 5/6 and the seven-pass transmembrane protein Frizzled (Fzd), which contains a Wnt-binding site in the form of a cysteine-rich domain. Known extracellular antagonists of the Wnt signaling pathway can be subdivided into two broad classes depending on whether they bind primarily to Wnt or to low-density lipoprotein receptor-related protein 5/6. We show that the secreted protein Tsukushi (TSK) functions as a Wnt signaling inhibitor by binding directly to the cysteine-rich domain of Fzd4 with an affinity of 2.3 × 10(-10) M and competing with Wnt2b. In the developing chick eye, TSK is expressed in the ciliary/iris epithelium, whereas Wnt2b is expressed in the adjacent anterior rim of the optic vesicle, where it controls the differentiation of peripheral eye structures, such as the ciliary body and iris. TSK overexpression effectively antagonizes Wnt2b signaling in chicken embryonic retinal cells both in vivo and in vitro and represses Wnt-dependent specification of peripheral eye fates. Conversely, targeted inactivation of the TSK gene in mice causes expansion of the ciliary body and up-regulation of Wnt2b and Fzd4 expression in the developing peripheral eye. Thus, we uncover a crucial role for TSK as a Wnt signaling inhibitor that regulates peripheral eye formation.

Equarin is involved in cell adhesion by means of heparan sulfate proteoglycan during lens development.

BACKGROUND: Adhesion molecules are known to be instructive for both development and differentiation. During lens differentiation, epithelial cells undergo vertical elongation, with the anterior and posterior tips of the elongating fiber cells sliding along the epithelium and capsule, respectively. These cellular processes are highly coordinated through cell adhesive interactions, actin cytoskeletal reorganization and contractile force generation. Alterations in extracellular matrix composition that interfere with these interactions can lead to defects that alter tissue morphogenesis and the state of differentiation. We have demonstrated that Equarin, which is a secreted molecule expressed in the equator region of the lens, plays an important role in chick lens fiber differentiation through fibroblast growth factor signaling. RESULTS: Here, we explored the function of Equarin in chick lens cell adhesion. Equarin protein was expressed in the extracellular region of lens differentiating cells. We found that Equarin promoted lens cell adhesion through heparan sulfate proteoglycan. By biochemical analysis, we found that Equarin directly binds syndecan-3, which displayed a similar expression pattern to Equarin. Overexpression of Equarin resulted in altered actin localization. CONCLUSIONS: Equarin is involved in cell adhesion during fiber differentiation and development.

Bacterial Ribosomes Induce Plasticity in Mouse Adult Fibroblasts.

The incorporation of bacterial ribosome has been reported to induce multipotency in somatic and cancer cells which leads to the conversion of cell lineages. Queried on its universality, we observed that bacterial ribosome incorporation into trypsinized mouse adult fibroblast cells (MAF) led to the formation of ribosome-induced cell clusters (RICs) that showed strong positive alkaline phosphatase staining. Under in vitro differentiation conditions, RICs-MAF were differentiated into adipocytes, osteoblasts, and chondrocytes. In addition, RICs-MAF were able to differentiate into neural cells. Furthermore, RICs-MAF expressed early senescence markers without cell death. Strikingly, no noticeable expression of renowned stemness markers like Oct4, Nanog, Sox2, etc. was observed here. Later RNA-sequencing data revealed the expression of rare pluripotency-associated markers, i.e., Dnmt3l, Sox5, Tbx3 and Cdc73 in RICs-MAF and the enrichment of endogenous ribosomal status. These observations suggested that RICs-MAF might have experienced a non-canonical multipotent state during lineage conversion. In sum, we report a unique approach of an exo-ribosome-mediated plastic state of MAF that is amenable to multi-lineage conversion.

Tsukushi controls the hair cycle by regulating TGF-ß1 signaling.

The hair follicle contains stem/progenitor cells that supply progeny for skin development and the hair cycle. Several signaling molecules belonging to the Wnt, BMP, shh, and transforming growth factor ß (TGF-ß) signaling cascades are involved in the normal hair follicle cycle. However, the systemic mechanism of how these humoral factors are controlled remains largely unknown. Previously, we reported that Tsukushi (TSK), a member of the small leucine-rich repeat proteoglycan family, functions extracellularly as a key coordinator of multiple signaling networks. Here, we show that TSK is expressed at the restricted areas of hair follicle during the morphogenesis and the hair cycle. Targeted disruption of the TSK gene causes the hair cycle to be delayed with low levels of TGF-ß1 and phosphorylated Smad2/3 (pSmad2/3) expression. Biochemical analysis indicates that TSK directly binds to TGF-ß1. Our data suggest that TSK controls the hair cycle by regulating TGF-ß1 signaling.

Equarin is involved as an FGF signaling modulator in chick lens differentiation.

Lens growth involves the proliferation of epithelial cells, followed by their migration to the equator region and differentiation into secondary fiber cells. It is widely accepted that fibroblast growth factor (FGF) signaling is required for the differentiation of lens epithelial cells into crystallin-rich fibers, but this signaling is insufficient to induce full differentiation. To better understand lens development, investigatory and functional analyses of novel molecules are required. Here, we demonstrate that Equarin, which is a novel secreted molecule, was expressed exclusively in the lens equator region during chick lens development. Equarin upregulated the expression of fiber markers, as demonstrated using in ovo electroporation. In a primary lens cell culture, Equarin promoted the biochemical and morphological changes associated with the differentiation of lens epithelial cells to fibers. A loss-of-function analysis was performed using zinc-finger nucleases targeting the Equarin gene. Lens cell differentiation was markedly inhibited when endogenous Equarin was blocked, indicating that Equarin was essential for normal chick lens differentiation. Furthermore, biochemical analysis showed that Equarin directly bound to FGFs and heparan sulfate proteoglycan and thereby upregulated the expression of phospho-ERK1/2 (ERK-P) proteins, the downstream of the FGF signaling pathway, in vivo and in vitro. Conversely, the absence of endogenous Equarin clearly diminished FGF-induced fiber differentiation. Taken together, our results suggest that Equarin is involved as an FGF modulator in chick lens differentiation.

Draxin inhibits axonal outgrowth through the netrin receptor DCC.

Draxin, a recently identified axon guidance protein, is essential for the formation of forebrain commissures, and can mediate repulsion of netrin-stimulated spinal commissural axons. Here, we report that draxin binds multiple netrin receptors: DCC (deleted in colorectal cancer), Neogenin, UNC5s (H1, H2, H3), and DSCAM (Down's syndrome cell adhesion molecule). Since draxin and Dcc knockouts showed similar phenotype in forebrain commissures formation, we show here the functional importance of draxin/DCC interaction. Draxin interacts with subnanomolar affinity to the netrin receptor DCC, in a region of DCC distinct from its netrin-binding domain. In vitro, neurite outgrowth from cortical and olfactory bulb explants of Dcc knock-out mice is significantly less inhibited by draxin, when compared with neurites from explants of wild-type mice. Furthermore, in comparison with wild-type mice, the growth cone collapse in response to draxin is largely abolished in Dcc-deficient cortical neurons. In vivo, double heteros of draxin/Dcc mice show markedly higher frequency of complete agenesis of corpus callosum than either of the single hetero. These results identify DCC as a convergent receptor for netrin and draxin in axon growth and guidance.