Takako Ooshio

TGF-β–FOXO signalling maintains leukaemia-initiating cells in chronic myeloid leukaemia

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-β is a critical regulator of Akt activation in LICs and controls Foxo3a localization. A combination of TGF-β inhibition, Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo. Furthermore, the treatment of human CML LICs with a TGF-β inhibitor impaired their colony-forming ability in vitro. Our results demonstrate a critical role for the TGF-β–FOXO pathway in the maintenance of LICs, and strengthen our understanding of the mechanisms that specifically maintain CML LICs in vivo.

Regulation of the Assembly and Adhesion Activity of E-cadherin by Nectin and Afadin for the Formation of Adherens Junctions in Madin-Darby Canine Kidney Cells

The Ca2+-independent immunoglobulin-like molecule nectin first forms cell-cell adhesion and then assembles cadherin at nectin-based cell-cell adhesion sites, resulting in the formation of adherens junctions (AJs). Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Here, we studied the roles and modes of action of nectin and afadin in the formation of AJs in cultured MDCK cells. The trans-interaction of nectin assembled E-cadherin, which associated with p120ctn, β-catenin, and α-catenin, at the nectin-based cell-cell adhesion sites in an afadin-independent manner. However, the assembled E-cadherin showed weak cell-cell adhesion activity and might be the non-trans-interacting form. This assembly was mediated by the IQGAP1-dependent actin cytoskeleton, which was organized by Cdc42 and Rac small G proteins that were activated by the action of trans-interacting nectin through c-Src and Rap1 small G protein in an afadin-independent manner. However, Rap1 bound to afadin, and this Rap1-afadin complex then interacted with p120ctn associated with non-trans-interacting E-cadherin, thereby causing the trans-interaction of E-cadherin. Thus, nectin regulates the assembly and cell-cell adhesion activity of E-cadherin through afadin, nectin signaling, and p120ctn for the formation of AJs in Madin-Darby canine kidney cells.

mTORC1 is essential for leukemia propagation but not stem cell self-renewal

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.

Cooperative roles of Par-3 and afadin in the formation of adherens and tight junctions

Par-3 is a cell-polarity protein that regulates the formation of tight junctions (TJs) in epithelial cells, where claudin is a major cell-cell adhesion molecule (CAM). TJs are formed at the apical side of adherens junctions (AJs), where E-cadherin and nectin are major CAMs. We have revealed that nectin first forms cell-cell adhesions, and then recruits cadherin to nectin-based cell-cell adhesion sites to form AJs and subsequently recruits claudin to the apical side of AJs to form TJs. The cytoplasmic tail of nectin binds afadin and Par-3. Afadin regulates the formation of AJs and TJs cooperatively with nectin. Here, we studied the role of Par-3 in the formation of these junctions by using Par-3-knockdown MDCK cells. Par-3 was necessary for the formation of AJs and TJs but was not necessary for nectin-based cell-cell adhesion. Par-3 promoted the association of afadin with nectin, whereas afadin was not necessary for the association of Par-3 with nectin. However, the association of afadin with nectin alone was not sufficient for the formation of AJs or TJs, and Par-3 and afadin cooperatively regulated it. We describe here these novel roles of Par-3 in the formation of junctional complexes.

Involvement of the Interaction of Afadin with ZO-1 in the Formation of Tight Junctions in Madin-Darby Canine Kidney Cells

Tight junctions (TJs) and adherens junctions (AJs) are major junctional apparatuses in epithelial cells. Claudins and junctional adhesion molecules (JAMs) are major cell adhesion molecules (CAMs) at TJs, whereas cadherins and nectins are major CAMs at AJs. Claudins and JAMs are associated with ZO proteins, whereas cadherins are associated with β- and α-catenins, and nectins are associated with afadin. We previously showed that nectins first form cell-cell adhesions where the cadherin-catenin complex is recruited to form AJs, followed by the recruitment of the JAM-ZO and claudin-ZO complexes to the apical side of AJs to form TJs. It is not fully understood how TJ components are recruited to the apical side of AJs. We studied the roles of afadin and ZO-1 in the formation of TJs in Madin-Darby canine kidney (MDCK) cells. Before the formation of TJs, ZO-1 interacted with afadin through the two proline-rich regions of afadin and the SH3 domain of ZO-1. During and after the formation of TJs, ZO-1 dissociated from afadin and associated with JAM-A. Knockdown of afadin impaired the formation of both AJs and TJs in MDCK cells, whereas knockdown of ZO-1 impaired the formation of TJs, but not AJs. Re-expression of full-length afadin restored the formation of both AJs and TJs in afadin-knockdown MDCK cells, whereas re-expression of afadin-ΔPR1–2, which is incapable of binding to ZO-1, restored the formation of AJs, but not TJs. These results indicate that the transient interaction of afadin with ZO-1 is necessary for the formation of TJs in MDCK cells.

Identification of tumor-initiating cells in a highly aggressive brain tumor using promoter activity of nucleostemin

Controversy remains over whether the cancer stem cell (CSC) theory applies to all tumors. To determine whether cells within a highly aggressive solid tumor are stochastically or hierarchically organized, we combined a reporter system where the nucleostemin (NS) promoter drives GFP expression (termed NS-GFP) with a mouse brain tumor model induced by retroviral Ras expression on a p16Ink4a/p19Arf-deficient background. The NS-GFP system allowed us to monitor the differentiation process of normal neural stem/precursor cells by analyzing GFP fluorescence intensity. In tumor-bearing mice, despite the very high frequency of tumorigenic cells, we successfully identified the NS-GFP+ cells as tumor-initiating cells (T-ICs). The clonal studies conclusively established that phenotypical heterogeneity can exist among the cells comprising a genetically homogeneous tumor, suggesting that this aggressive brain tumor follows the CSC model. Detailed analyses of the NS-GFP+ brain tumor cells revealed that T-ICs showed activation of the receptor tyrosine kinase c-Met, which functions in tumor invasiveness. Thus, the NS-GFP system provides a powerful tool to elucidate stem cell biology in normal and malignant tissues.

Requirement of the actin cytoskeleton for the association of nectins with other cell adhesion molecules at adherens and tight junctions in MDCK cells.

Nectins, Ca2+‐independent immunoglobulin‐like cell adhesion molecules (CAMs), first form cell‐cell adhesion where cadherins are recruited, forming adherens junctions (AJs) in epithelial cells and fibroblasts. In addition, nectins recruit claudins, occludin, and junctional adhesion molecules (JAMs) to the apical side of AJs, forming tight junctions (TJs) in epithelial cells. Nectins are associated with these CAMs through peripheral membrane proteins (PMPs), many of which are actin filament‐binding proteins. We examined here the roles of the actin cytoskeleton in the association of nectins with other CAMs in MDCK cells stably expressing exogenous nectin‐1. The nectin‐1‐based cell‐cell adhesion was formed and maintained irrespective of the presence and absence of the actin filament‐disrupting agents, such as cytochalasin D and latrunculin A. In the presence of these agents, only afadin remained at the nectin‐1‐based cell‐cell adhesion sites, whereas E‐cadherin and other PMPs at AJs, α‐catenin, β‐catenin, vinculin, α‐actinin, ADIP, and LMO7, were not concentrated there. The CAMs at TJs, claudin‐1, occludin and JAM‐1, or the PMPs at TJs, ZO‐1 and MAGI‐1, were not concentrated there, either. These results indicate that the actin cytoskeleton is required for the association of the nectin‐afadin unit with other CAMs and PMPs at AJs and TJs.

Requirement of nectin, but not cadherin, for formation of claudin-based tight junctions in annexin II-knockdown MDCK cells

Adherens junctions (AJs) and tight junctions (TJs) comprise a junctional complex which plays key roles not only in cell adhesion and polarization but also in regulation of cell movement and proliferation in epithelial cells. E-Cadherin and nectin are major cell–cell adhesion molecules (CAMs) at AJs, whereas claudin is a major CAM at TJs. We have shown that the cadherin-based cell–cell adhesion is not formed in MDCK cells in which annexin II, a Ca2+- and phospholipid-binding protein, is knocked down. Here, we found that TJs and the nectin-based cell–cell adhesions were formed in annexin II-knockdown cells. The formation of TJs in annexin II-knockdown MDCK cells required the nectin-based cell–cell adhesion and afadin, a nectin- and actin-filament-binding protein. In addition, it required the activation of Cdc42 and Rac small G proteins and subsequent reorganization of the IQGAP1-dependent actin cytoskeleton which were induced by the nectin-based cell–cell adhesion. These results indicate that the nectin-based cell–cell adhesion and afadin, but not the cadherin-based cell–cell adhesion, are necessary for the formation of TJs and that the signaling by nectin and the subsequent reorganization of the actin cytoskeleton are also necessary for the formation of TJs under certain conditions.

Identification of Stem Cells During Prepubertal Spermatogenesis via Monitoring of Nucleostemin Promoter Activity

The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S‐phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS‐GFP Tg mice). In adult NS‐GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS‐GFP Tg embryos, neonates, and 10‐day‐old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long‐term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell‐cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis.

Involvement of heterophilic trans-interaction of Necl-5/Tage4/PVR/CD155 with nectin-3 in formation of nectin- and cadherin-based adherens junctions

Nectins, Ca2+‐independent immunoglobulin (Ig)‐like cell–cell adhesion molecules and cadherins, Ca2+‐dependent cell–cell adhesion molecules, are associated through their respective cytoplasmic tail‐binding proteins, afadin and catenins and play roles in formation of adherens junctions (AJs) in epithelial cells and fibroblasts. Nectin‐like molecule‐5 (Necl‐5) is a Ca2+‐independent Ig‐like molecule which does not homophilically trans‐interact, but heterophilically trans‐interacts with nectin‐3, one member of the nectin family. Necl‐5 does not directly bind afadin and therefore is not associated with cadherins. Necl‐5 regulates cell motility and proliferation in cooperation with integrins and growth factor receptors, when it does not interact with nectin‐3. We studied here a role of the heterophilic trans‐interaction of Necl‐5 with nectin‐3 in cell–cell adhesion using L cells stably expressing Necl‐5, nectin‐3 and E‐cadherin (Necl‐5‐nectin‐3‐EL cells). Afadin, E‐cadherin and catenins were recruited to the nectin‐3 side, but not to the Necl‐5 side, of the contact sites formed by the heterophilic trans‐interaction between Necl‐5 and nectin‐3. The anti‐Necl‐5 monoclonal antibody, which specifically inhibited the heterophilic trans‐interaction of Necl‐5 with nectin‐3, inhibited the formation of the E‐cadherin‐based AJs in Necl‐5‐nectin‐3‐EL cells. These results indicate that Necl‐5 plays roles not only in cell motility and proliferation but also in cell–cell adhesion in cooperation with nectin‐3.