[Show abstract][Hide abstract] ABSTRACT: The Notch signal regulates both cell viability and apoptosis, and maintains stemness of various cancers including glioblastoma (GBM). Although Notch signal inhibition may be an effective strategy in treating GBM initiating cells (GICs), its applicability to the different subtypes of GBM remains unclear. Here, we analyzed the effectiveness of MRK003, a preclinical γ-secretase inhibitor, on GICs. Nine patient-derived GICs were treated by MRK003, and its efficacy on cell viability, apoptosis, sphere forming ability and Akt expression level which might be related to Notch downstream and be greatly important signals in GBM was evaluated. MRK003 suppressed viability and sphere-formation ability, and induced apoptosis in all GICs in varying doses of MRK003. Based on their sensitivities to MRK003, the nine GICs were divided into "relatively sensitive" and "relatively resistant" GICs. Sensitivity to MRK003 was associated with its inhibitory effect on Akt pathway. Transgenic expression of the myristoylated Akt vector in relatively sensitive GICs partially rescued the effect of MRK003, suggesting that the effect of MRK003 was, at least in part, mediated through inhibition of the Akt pathway. These GICs were differentiated by the expression of CD44 and CD133 with flow cytometric analysis. The relatively sensitive GICs are CD44-high and CD133-low. The IC50 of MRK003 in a set of GICs exhibited a negative correlation with CD44 and positive correlation with CD133. Collectively, MRK003 is partially mediated by the Akt pathway and has strong therapeutic potential for CD44-high and CD133-low GICs.
Journal of Neuro-Oncology 10/2014; · 3.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acute myeloid leukaemia (AML) is a heterogeneous neoplastic disorder in which a subset of cells function as leukaemia-initiating cells (LICs). In this study, we prospectively evaluated the leukaemia-initiating capacity of AML cells fractionated according to the expression of a nucleolar GTP binding protein, nucleostemin (NS). To monitor NS expression in living AML cells, we generated a mouse AML model in which green fluorescent protein (GFP) is expressed under the control of a region of the NS promoter (NS-GFP). In AML cells, NS-GFP levels were correlated with endogenous NS mRNA. AML cells with the highest expression of NS-GFP were very immature blast-like cells, efficiently formed leukaemia colonies in vitro, and exhibited the highest leukaemia-initiating capacity in vivo. Gene expression profiling analysis revealed that cell cycle regulators and nucleotide metabolism-related genes were highly enriched in a gene set associated with leukaemia-initiating capacity that we termed the 'leukaemia stem cell gene signature'. This gene signature stratified human AML patients into distinct clusters that reflected prognosis, demonstrating that the mouse leukaemia stem cell gene signature is significantly associated with the malignant properties of human AML. Further analyses of gene regulation in leukaemia stem cells could provide novel insights into diagnostic and therapeutic approaches to AML.
Biochemical and Biophysical Research Communications 06/2014; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: mTOR is an evolutionarily conserved kinase that plays a critical role in sensing and responding to environmental determinants. Recent studies have shown that fine-tuning of the activity of mTOR complexes contributes to organogenesis and tumorigenesis. Although rapamycin, an allosteric mTOR inhibitor, is an effective immunosuppressant, the precise roles of mTOR complexes in early T-cell development remain unclear. Here we show that mTORC1 plays a critical role in the development of both early T-cell progenitors and leukemia. Deletion of Raptor, an essential component of mTORC1, produced defects in the earliest development of T-cell progenitors in vivo and in vitro. Deficiency of Raptor resulted in cell cycle abnormalities in early T-cell progenitors that were associated with instability of the Cyclin D2/D3-CDK6 complexes; deficiency of Rictor, an mTORC2 component, did not have the same effect, indicating that mTORC1 and -2 control T-cell development in different ways. In a model of myeloproliferative neoplasm and T-cell acute lymphoblastic leukemia (T-ALL) evoked by Kras activation, Raptor deficiency dramatically inhibited the cell cycle in oncogenic Kras-expressing T-cell progenitors, but not myeloid progenitors, and specifically prevented the development of T-ALL. Although rapamycin treatment significantly prolonged the survival of recipient mice bearing T-ALL cells, rapamycin-insensitive leukemia cells continued to propagate in vivo. In contrast, Raptor deficiency in the T-ALL model resulted in cell cycle arrest and efficient eradication of leukemia. Thus, understanding the cell-context-dependent role of mTORC1 illustrates the potential importance of mTOR signals as therapeutic targets.
Proceedings of the National Academy of Sciences 02/2014; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glioblastomas frequently harbor genetic lesions that stimulate the activity of mammalian target of rapamycin complex 1 (mTORC1). Loss of heterozygosity of tuberous sclerosis complex 1 (TSC1) or TSC2, which together form a critical negative regulator of mTORC1, is also seen in glioblastoma; however, it is not known how loss of the TSC complex affects the development of malignant gliomas. Here we investigated the role of Tsc1 in gliomagenesis in mice. Tsc1 deficiency upregulated mTORC1 activity and suppressed the proliferation of neural stem/progenitor cells (NSPCs) in a serial neurosphere-forming assay, suggesting that Tsc1-deficient NSPCs have defective self-renewal activity. The neurosphere-forming capacity of Tsc1-deficient NSPCs was restored by p16(Ink4a)p19(Arf) deficiency. Combined Tsc1 and p16(Ink4a)p19(Arf) deficiency in NSPCs did not cause gliomagenesis in vivo. However, in a glioma model driven by an active mutant of EGFR, EGFRvIII, loss of Tsc1 resulted in earlier onset of glioma development. The mTORC1 hyperactivation by Tsc1 deletion accelerated malignant phenotypes, including increased tumor mass and enhanced microvascular formation leading to intracranial hemorrhage. These data demonstrate that, although mTORC1 hyperactivation itself may not be sufficient for gliomagenesis, it is a potent modifier of glioma development when combined with oncogenic signals.
Journal of Biochemistry 12/2013; · 3.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nucleostemin (NS) is a nucleolar GTP-binding protein that is involved in ribosomal biogenesis and protection of telomeres. We investigated the expression of NS in human germ cell tumors and its function in a mouse germ cell tumor model. NS was abundantly expressed in undifferentiated, but not differentiated, types of human testicular germ cell tumors. NS was expressed concomitantly with OCT3/4, a critical regulator of the undifferentiated status of pluripotent stem cells in primordial germ cells and embryonal carcinomas. To investigate the roles of NS in tumor growth in vivo, we used a mouse teratoma model. Analysis of teratomas derived from embryonic stem cells in which the NS promoter drives GFP expression showed that cells highly expressing NS were actively proliferating and exhibited the characteristics of tumor-initiating cells, including the ability to initiate and propagate tumor cells in vivo. NS-expressing cells exhibited higher levels of GTP than non-NS-expressing cells. Because NS protein is stabilized by intracellular GTP, metabolic changes may contribute to abundant NS expression in the undifferentiated cells. OCT3/4 deficiency in teratomas led to loss of NS expression, resulting in growth retardation. Finally, we found that teratomas deficient in NS lost their undifferentiated characteristics, resulting in defective tumor proliferation. These data indicate that abundant expression of NS supports the undifferentiated properties of germ cell tumors.
American Journal Of Pathology 08/2013; 183(2):592-603. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The high regenerative capacity of liver contributes to the maintenance of its size and function when injury occurs. Partial hepatectomy induces division of mature hepatocytes to maintain liver function, whereas severe injury stimulates expansion of undifferentiated hepatic precursor cells, which supply mature cells. Although several factors reportedly function in liver regeneration, the precise mechanisms underlying regeneration remain unclear. In this study, we analyzed expression of nucleostemin (NS) during development and in injured liver by using transgenic green fluorescent protein reporter (NS-GFP Tg) mice. In neonatal liver, the hepatic precursor cells that give rise to mature hepatocytes were enriched in a cell population expressing high levels of NS. In adult liver, NS was abundantly expressed in mature hepatocytes and rapidly upregulated by partial hepatectomy. Severe liver injury promoted by a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine induced the emergence of NS-expressing ductal epithelial cells as hepatic precursor cells. NS knockdown inhibited both hepatic colony formation in vitro and proliferation of hepatocytes in vivo. These data strongly suggest that NS plays a critical role in regeneration of both hepatic precursor cells and hepatocytes in response to liver injury.
Stem cells and development 07/2012; 21(16):3044-54. · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although dysregulation of mTOR complex 1 (mTORC1) promotes leukemogenesis, how mTORC1 affects established leukemia is unclear. We investigated the role of mTORC1 in mouse hematopoiesis using a mouse model of conditional deletion of Raptor, an essential component of mTORC1. Raptor deficiency impaired granulocyte and B cell development but did not alter survival or proliferation of hematopoietic progenitor cells. In a mouse model of acute myeloid leukemia (AML), Raptor deficiency significantly suppressed leukemia progression by causing apoptosis of differentiated, but not undifferentiated, leukemia cells. mTORC1 did not control cell cycle or cell growth in undifferentiated AML cells in vivo. Transplantation of Raptor-deficient undifferentiated AML cells in a limiting dilution revealed that mTORC1 is essential for leukemia initiation. Strikingly, a subset of AML cells with undifferentiated phenotypes survived long-term in the absence of mTORC1 activity. We further demonstrated that the reactivation of mTORC1 in those cells restored their leukemia-initiating capacity. Thus, AML cells lacking mTORC1 activity can self-renew as AML stem cells. Our findings provide mechanistic insight into how residual tumor cells circumvent anticancer therapies and drive tumor recurrence.
The Journal of clinical investigation 05/2012; 122(6):2114-29. · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent improvements in cell purification and transplantation techniques have contributed to the identification of cell populations known as tumor-initiating cells (TIC). This discovery has led to the 'cancer stem cell hierarchy' concept, which holds that tumors are organized as a hierarchy of malignant tissues sustained by such TIC. However, this concept remains controversial. In this review, we examine recent advances in cancer stem cell research that have been generated from studies of Philadelphia (Ph) chromosome-positive leukemia. The abnormal Ph chromosome, which arises from a translocation creating the BCR-ABL1 fusion gene, is most commonly associated with chronic myelogenous leukemia (CML) and precursor B cell acute lymphoblastic leukemia (B-ALL). Examination of the pathophysiology of these diseases has provided interesting insights into not only the hierarchy of leukemia stem cells but also their clonal evolution. Both shared and unique regulatory mechanisms affecting normal and CML stem cell behavior have been identified. On the other hand, genetic diversity in specific clones of Ph(+) B-ALL that drive clonal evolution has recently come to light. Our expanding knowledge of the biology of TIC contributes to the progress of cancer research, and may open the door to new concepts in cancer therapy.
Pathology International 09/2011; 61(9):501-8. · 1.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In most stem cell systems, the organization of the stem cell niche and the anchoring matrix required for stem cell maintenance are largely unknown. We report here that collagen XVII (COL17A1/BP180/BPAG2), a hemidesmosomal transmembrane collagen, is highly expressed in hair follicle stem cells (HFSCs) and is required for the maintenance not only of HFSCs but also of melanocyte stem cells (MSCs), which do not express Col17a1 but directly adhere to HFSCs. Mice lacking Col17a1 show premature hair graying and hair loss. Analysis of Col17a1-null mice revealed that COL17A1 is critical for the self-renewal of HFSCs through maintaining their quiescence and immaturity, potentially explaining the mechanism underlying hair loss in human COL17A1 deficiency. Moreover, forced expression of COL17A1 in basal keratinocytes, including HFSCs, in Col17a1-null mice rescues MSCs from premature differentiation and restores TGF-β signaling, demonstrating that HFSCs function as a critical regulatory component of the MSC niche.
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma (GBM) is the most aggressive and destructive form of brain cancer. Animal models that can unravel the mechanisms underlying its progression are needed to develop rational and effective molecular therapeutic approaches. In this study, we report the development of mouse models for spontaneous gliomas representing distinct progressive stages of disease that are governed by defined genetic alterations. Neural stem/progenitor cell (NPC)-specific constitutive Ras activation in vivo plus p53 deficiency led to development of primarily anaplastic astrocytoma (grade III), whereas combined loss of p53 plus p16(Ink4a)/p19(Arf) led to development of GBM (grade IV) at 100% penetrance within 6 weeks. These glioma models showed enhanced stem cell properties (stemness) accompanied by malignant progression. Notably, we determined that, in our models and in human specimens, downregulation of the homeodomain transcription factor NKX2.2, which is essential for oligodendroglial differentiation, was correlated with increased tumor malignancy. NKX2.2 overexpression by GBM-derived glioma-initiating cells (GIC) induced oligodendroglial differentiation and suppressed self-renewal capacity. By contrast, Nkx2.2 downregulation in mouse NPCs accelerated GBM formation. Importantly, the inhibitory effects of NXK2.2 on GIC self-renewal were conserved in human cells. Thus, our mouse models offer pathobiologically significant advantages to investigate the nature of brain tumors, with improved opportunities to develop novel mechanism-based therapeutic approaches.
Cancer Research 02/2011; 71(3):1135-45. · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic myeloid leukaemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase. It is widely believed that BCR-ABL activates Akt signalling that suppresses the forkhead O transcription factors (FOXO), supporting the proliferation or inhibiting the apoptosis of CML cells. Although the use of the tyrosine kinase inhibitor imatinib is a breakthrough for CML therapy, imatinib does not deplete the leukaemia-initiating cells (LICs) that drive the recurrence of CML. Here, using a syngeneic transplantation system and a CML-like myeloproliferative disease mouse model, we show that Foxo3a has an essential role in the maintenance of CML LICs. We find that cells with nuclear localization of Foxo3a and decreased Akt phosphorylation are enriched in the LIC population. Serial transplantation of LICs generated from Foxo3a(+/+) and Foxo3a(-/-) mice shows that the ability of LICs to cause disease is significantly decreased by Foxo3a deficiency. Furthermore, we find that TGF-beta is a critical regulator of Akt activation in LICs and controls Foxo3a localization. A combination of TGF-beta inhibition, Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo. Furthermore, the treatment of human CML LICs with a TGF-beta inhibitor impaired their colony-forming ability in vitro. Our results demonstrate a critical role for the TGF-beta-FOXO pathway in the maintenance of LICs, and strengthen our understanding of the mechanisms that specifically maintain CML LICs in vivo.
[Show abstract][Hide abstract] ABSTRACT: DNA methylation is an epigenetic modification essential for development. The DNA methyltransferases Dnmt3a and Dnmt3b execute de novo DNA methylation in gastrulating embryos and differentiating germline cells. It has been assumed that these enzymes generally play a role in regulating cell differentiation. To test this hypothesis, we examined the role of Dnmt3a and Dnmt3b in adult stem cells. CD34(-/low), c-Kit(+), Sca-1(+), lineage marker(-) (CD34(-) KSL) cells, a fraction of mouse bone marrow cells highly enriched in hematopoietic stem cells (HSCs), expressed both Dnmt3a and Dnmt3b. Using retroviral Cre gene transduction, we conditionally disrupted Dnmt3a, Dnmt3b, or both Dnmt3a and Dnmt3b (Dnmt3a/Dnmt3b) in CD34(-) KSL cells purified from mice in which the functional domains of these genes are flanked by two loxP sites. We found that Dnmt3a and Dnmt3b function as de novo DNA methyltransferases during differentiation of hematopoietic cells. Unexpectedly, in vitro colony assays and in vivo transplantation assays showed that both myeloid and lymphoid lineage differentiation potentials were maintained in Dnmt3a-, Dnmt3b-, and Dnmt3a/Dnmt3b-deficient HSCs. However, Dnmt3a/Dnmt3b-deficient HSCs, but not Dnmt3a- or Dnmt3b-deficient HSCs, were incapable of long-term reconstitution in transplantation assays. These findings establish a critical role for DNA methylation by Dnmt3a and Dnmt3b in HSC self-renewal.
Journal of Experimental Medicine 05/2007; 204(4):715-22. · 13.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: One of the central tasks of stem cell biology is to understand the molecular mechanisms that control self-renewal in stem cells. Several cytokines are implicated as crucial regulators of hematopoietic stem cells (HSCs), but little is known about intracellular signaling for HSC self-renewal. To address this issue, we attempted to clarify how self-renewal potential is enhanced in HSCs without the adaptor molecule Lnk, as in Lnk-deficient mice HSCs are expanded in number >10-fold because of their increased self-renewal potential. We show that Lnk negatively regulates self-renewal of HSCs by modifying thrombopoietin (TPO)-mediated signal transduction. Single-cell cultures showed that Lnk-deficient HSCs are hypersensitive to TPO. Competitive repopulation revealed that long-term repopulating activity increases in Lnk-deficient HSCs, but not in WT HSCs, when these cells are cultured in the presence of TPO with or without stem cell factor. Single-cell transplantation of each of the paired daughter cells indicated that a combination of stem cell factor and TPO efficiently induces symmetrical self-renewal division in Lnk-deficient HSCs but not in WT HSCs. Newly developed single-cell immunostaining demonstrated significant enhancement of both p38 MAPK inactivation and STAT5 and Akt activation in Lnk-deficient HSCs after stimulation with TPO. Our results suggest that a balance in positive and negative signals downstream from the TPO signal plays a role in the regulation of the probability of self-renewal in HSCs. In general, likewise, the fate of stem cells may be determined by combinational changes in multiple signal transduction pathways.
Proceedings of the National Academy of Sciences 02/2007; 104(7):2349-54. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mouse hematopoietic stem cells (HSCs) are the best-studied stem cells because functional assays for mouse HSCs were established earliest and purification techniques for mouse HSCs have progressed furthest. Here we describe our current protocols for the purification of CD34-
KSL) cells, the HSC population making up approximately 0.005% of bone marrow cells in adult C557BL/6 mice. Purified HSCs have been characterized at cellular and molecular levels. Since clonal analysis is essential for the study of self-renewal and lineage commitment in HSCs, here we present our single-cell colony assay and single-cell transplantation procedures. We also introduce our immunostaining procedures for small numbers of HSCs, which are useful for signal transduction analysis. The purification of CD34-
KSL cells requires approximately 6 h. Initialization of single-cell culture requires approximately 1 h. Single-cell transplantation requires approximately 6 h. Single-cell immunostaining requires approximately 2 d.
[Show abstract][Hide abstract] ABSTRACT: Although the concept of a leukemic stem cell system has recently been well accepted, its nature and the underlying molecular mechanisms remain obscure. Constitutive activation of signal transducers and activators of transcription 3 (STAT3) and STAT5 is frequently detected in various hematopoietic tumors. To evaluate their role in normal and leukemic stem cells, we took advantage of constitutively active STAT mutants to activate STAT signaling selectively in hematopoietic stem cells (HSCs). Activation of STAT5 in CD34- c-Kit+ Sca-1+ lineage marker- (CD34- KSL) HSCs led to a drastic expansion of multipotential progenitors and promoted HSC self-renewal ex vivo. In sharp contrast, STAT3 was demonstrated to be dispensable for the HSC maintenance in vivo, and its activation facilitated lineage commitment of HSCs in vitro. In a mouse model of myeloproliferative disease (MPD), sustained STAT5 activation in CD34- KSL HSCs but not in CD34+ KSL multipotential progenitors induced fatal MPD, indicating that the capacity of STAT5 to promote self-renewal of hematopoietic stem cells is crucial to MPD development. Our findings collectively establish a specific role for STAT5 in self-renewal of normal as well as leukemic stem cells.
Journal of Experimental Medicine 08/2005; 202(1):169-79. · 13.21 Impact Factor