Cell-matrix interface regulates dormancy in human colon cancer stem cells.

Cancer relapse after chemotherapy remains a main cause of cancer-related death. Although the relapse is thought to result from the propagation of resident cancer stem cells1, a lack of experimental platforms that enable the prospective analysis of cancer stem cell dynamics with sufficient spatiotemporal resolution has hindered the testing of this hypothesis. Here we develop a live genetic lineage-tracing system that allows the longitudinal tracking of individual cells in xenotransplanted human colorectal cancer organoids, and identify LGR5+ cancer stem cells that exhibit a dormant behaviour in a chemo-naive state. Dormant LGR5+ cells are marked by the expression of p27, and intravital imaging provides direct evidence of the persistence of LGR5+p27+ cells during chemotherapy, followed by clonal expansion. Transcriptome analysis reveals that COL17A1-a cell-adhesion molecule that strengthens hemidesmosomes-is upregulated in dormant LGR5+p27+ cells. Organoids in which COL17A1 is knocked out lose the dormant LGR5+p27+ subpopulation and become sensitive to chemotherapy, which suggests that the cell-matrix interface has a role in the maintenance of dormancy. Chemotherapy disrupts COL17A1 and breaks the dormancy in LGR5+p27+ cells through FAK-YAP activation. Abrogation of YAP signalling prevents chemoresistant cells from exiting dormancy and delays the regrowth of tumours, highlighting the therapeutic potential of YAP inhibition in preventing cancer relapse. These results offer a viable therapeutic approach to overcome the refractoriness of human colorectal cancer to conventional chemotherapy.

Diastereoselective Synthesis of trans-anti-Hydrophenanthrenes via Ti-mediated Radical Cyclization and Total Synthesis of Kamebanin.

Ent-kaurenes consist of an ABC-ring based on a trans-anti-hydrophenanthrene skeleton and a D ring with an exomethylene. Highly oxygen-functionalized ent-kauren-15-ones have promising antiinflammatory pharmacological activity. In this study, we developed a novel diastereoselective synthesis of trans-anti-hydrophenanthrenes via a Ti-mediated reductive radical cyclization. We also demonstrated the applicability of this method by developing the first total synthesis of (±)-kamebanin (longest linear sequence; 17 steps, overall yield; 6.5 %). Furthermore, this synthesis provided a formal semi-pinacol rearrangement for the construction of the quaternary carbon at C8 and a novel Thorpe-Ziegler-type reaction for the construction of the D-ring.

Biomimetic Total Syntheses of (+)-Chloropupukeananin, (-)-Chloropupukeanolide D, and Chloropestolides.

Chloropupukeananin, chloropupukeanolides, and chloropestolides are a family of structurally complex bioactive natural products that possess highly functionalized tricyclo[4.3.1.03,7]decane or bicyclo[2.2.2]octane skeletons. Biosynthesis of the chloropupukeananin family is triggered by the intermolecular heterodimeric Diels-Alder reaction between maldoxin and iso-A82775C; however, the enzymes involved have not yet been identified. We herein report the one-pot biomimetic synthesis of chloropupukeananin and chloropupukeanolide D. Moreover, the effect of the solvent on the intermolecular Diels-Alder reaction of siccayne and maldoxin suggested that the biosynthesis of the chloropupukeananin family involves a Diels-Alderase-catalyzed heterodimeric Diels-Alder reaction.

Blockade of the fractalkine-CX3CR1 axis ameliorates experimental colitis by dislodging venous crawling monocytes.

Chemokine systems modulate inflammatory and immune responses in inflammatory bowel disease (IBD). The colons of IBD patients show increased levels of fractalkine (FKN) and high numbers of FKN receptor-positive (CX3CR1+) cells; however, the FKN-CX3CR1 axis's role in intestinal inflammation, especially in intravascular leukocyte behaviors, still remains unclear. Here, we show that interruption of the FKN-CX3CR1 axis by anti-FKN monoclonal antibody (mAb) ameliorates murine colitis through regulation of intravascular monocyte behaviors in murine colitis models. FKN expression was detectable in vascular endothelium and CX3CR1+ macrophages accumulated in the mucosal lamina propria and submucosa of the inflamed colons. CD115+ monocytes tethered to the venous endothelium and expressed pro-inflammatory mediators. The anti-FKN mAb improved colitis symptoms, markedly reduced pro-inflammatory factors in the colon, maintained blood vessel integrity and reduced tethered monocytes in the inflamed veins. Intravital imaging revealed that CD115+Gr-1low/- monocytes crawled on the apical surfaces of venous endothelium, and anti-FKN mAb rapidly dislodged the crawling monocytes and inhibited their patrolling behavior. These findings suggest that the FKN-CX3CR1 axis triggers the patrolling behavior of crawling monocytes on the venous endothelium of inflamed colons, and accelerates the subsequent leukocyte activation and infiltration by locally producing inflammatory cytokines and chemokines. The mAb also ameliorated symptoms in another IBD model, T-cell-transferred colitis. Blocking the FKN-CX3CR1 axis with an anti-FKN mAb considerably inhibits the colitis-triggered inflammatory cascades, which may be an alternative strategy to treat IBD.

Nectins and nectin-like molecules: roles in contact inhibition of cell movement and proliferation.

Nectins and nectin-like molecules (Necls) are immunoglobulin-like transmembrane cell adhesion molecules that are expressed in various cell types. Homophilic and heterophilic engagements between family members provide cells with molecular tools for intercellular communications. Nectins primarily regulate cell-cell adhesions, whereas Necls are involved in a greater variety of cellular functions. Recent studies have revealed that nectins and NECL-5, in cooperation with integrin alphavbeta3 and platelet-derived growth factor receptor, are crucial for the mechanisms that underlie contact inhibition of cell movement and proliferation; this has important implications for the development and tissue regeneration of multicellular organisms and the phenotypes of cancer cells.

Binding between the junctional proteins afadin and PLEKHA7 and implication in the formation of adherens junction in epithelial cells.

Adherens junction (AJ) is a specialized cell-cell junction structure that plays a role in mechanically connecting adjacent cells to resist strong contractile forces and to maintain tissue structure, particularly in the epithelium. AJ is mainly comprised of cell adhesion molecules cadherin and nectin and their associating cytoplasmic proteins including ß-catenin, α-catenin, p120(ctn), and afadin. Our series of studies have revealed that nectin first forms cell-cell adhesion and then recruits cadherin to form AJ. The recruitment of cadherin by nectin is mediated by the binding of α-catenin and p120(ctn) to afadin. Recent studies showed that PLEKHA7 binds to p120(ctn), which is associated with E-cadherin, and maintains the integrity of AJ in epithelial cells. In this study, we showed that PLEKHA7 bound to afadin in addition to p120(ctn) and was recruited to the nectin-3α-based cell-cell adhesion site in a manner dependent on afadin, but not on p120(ctn). The binding of PLEKHA7 to afadin was required for the proper formation of AJ, but not for the formation of tight junction, in EpH4 mouse mammary gland epithelial cells. These results indicate that PLEKHA7 plays a cooperative role with nectin and afadin in the proper formation of AJ in epithelial cells.

Nectin and junctional adhesion molecule are critical cell adhesion molecules for the apico-basal alignment of adherens and tight junctions in epithelial cells.

Tight junctions (TJs) and adherens junctions (AJs) form an apical junctional complex at the apical side of the lateral membranes of epithelial cells, in which TJs are aligned at the apical side of AJs. Many cell adhesion molecules (CAMs) and cell polarity molecules (CPMs) cooperatively regulate the formation of the apical junctional complex, but the mechanism for the alignment of TJs at the apical side of AJs is not fully understood. We developed a cellular system with which epithelial-like TJs and AJs were reconstituted in fibroblasts and analyzed the cooperative roles of CAMs and CPMs. We exogenously expressed various combinations of CAMs and CPMs in fibroblasts that express negligible amounts of these molecules endogenously. In these cells, the nectin-based cell-cell adhesion was formed at the apical side of the junctional adhesion molecule (JAM)-based cell-cell adhesion, and cadherin and claudin were recruited to the nectin-3- and JAM-based cell-cell adhesion sites to form AJ-like and TJ-like domains, respectively. This inversed alignment of the AJ-like and TJ-like domains was reversed by complementary expression of CPMs Par-3, atypical protein kinase C, Par-6, Crb3, Pals1 and Patj. We describe the cooperative roles of these CAMs and CPMs in the apico-basal alignment of TJs and AJs in epithelial cells.

The roles of nectins in cell adhesions: cooperation with other cell adhesion molecules and growth factor receptors.

Nectins are Ca(2+)-independent Ig-like cell adhesion molecules (CAMs) which homophilically and heterophilically interact in trans with nectins and form cell-cell adhesion. This cell-cell adhesion is involved in the formation of many types of cell-cell junctions such as adherens junctions, tight junctions, and synaptic junctions, cooperatively with other CAMs such as cadherins and claudins. Nectins transduce signals cooperatively with integrin alpha(v)beta(3), and regulate formation of cell-cell junctions. In addition, nectin interacts in cis with PDGF receptor and regulates its signaling for anti-apoptosis. Furthermore, nectin interacts in trans with nectin-like molecule-5 (Necl-5) and regulate cell movement and proliferation. We describe cooperative roles of nectins with other CAMs and growth factor receptors.

Live Cell Monitoring of Separase Activity, a Key Enzymatic Reaction for Chromosome Segregation, with Chimeric FRET-Based Molecular Sensor upon Cell Cycle Progression.

Separase is a key cysteine protease in the separation of sister chromatids through the digestion of the cohesin ring that inhibits chromosome segregation as a trigger of the metaphase-anaphase transition in eukaryotes. Its activity is highly regulated by binding with securin and cyclinB-CDK1 complex. These bindings prevent the proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties in biochemical examinations due to the instability of separase in vitro and the fact that few spatiotemporal resolution approaches exist for monitoring live separase activity throughout mitotic processes. Here, we have developed FRET-based molecular sensors, including GFP variants, with separase-cleavable sequences as donors and covalently attached fluorescent dyes as acceptor molecules. These are applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated the performance of equivalent molecular sensors, either localized or not localized inside the nucleus under cell cycle control, using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems that can be used specifically or statistically, which could lead to the elucidation of separase properties in detail.

Anti-CX3CL1 (fractalkine) monoclonal antibody attenuates lung and skin fibrosis in sclerodermatous graft-versus-host disease mouse model.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular injury and inflammation, followed by excessive fibrosis of the skin and other internal organs, including the lungs. CX3CL1 (fractalkine), a chemokine expressed on endothelial cells, supports the migration of macrophages and T cells that express its specific receptor CX3CR1 into targeted tissues. We previously reported that anti-CX3CL1 monoclonal antibody (mAb) treatment significantly inhibited transforming growth factor (TGF)-ß1-induced expression of type I collagen and fibronectin 1 in human dermal fibroblasts. Additionally, anti-mouse CX3CL1 mAb efficiently suppressed skin inflammation and fibrosis in bleomycin- and growth factor-induced SSc mouse models. However, further studies using different mouse models of the complex immunopathology of SSc are required before the initiation of a clinical trial of CX3CL1 inhibitors for human SSc. METHODS: To assess the preclinical utility and functional mechanism of anti-CX3CL1 mAb therapy in skin and lung fibrosis, a sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) mouse model was analyzed with immunohistochemical staining for characteristic infiltrating cells and RNA sequencing assays. RESULTS: On day 42 after bone marrow transplantation, Scl-cGVHD mice showed increased serum CX3CL1 level. Intraperitoneal administration of anti-CX3CL1 mAb inhibited the development of fibrosis in the skin and lungs of Scl-cGVHD model, and did not result in any apparent adverse events. The therapeutic effects were correlated with the number of tissue-infiltrating inflammatory cells and α-smooth muscle actin (α-SMA)-positive myofibroblasts. RNA sequencing analysis of the fibrotic skin demonstrated that cGVHD-dependent induction of gene sets associated with macrophage-related inflammation and fibrosis was significantly downregulated by mAb treatment. In the process of fibrosis, mAb treatment reduced cGVHD-induced infiltration of macrophages and T cells in the skin and lungs, especially those expressing CX3CR1. CONCLUSIONS: Together with our previous findings in other SSc mouse models, the current results indicated that anti-CX3CL1 mAb therapy could be a rational therapeutic approach for fibrotic disorders, such as human SSc and Scl-cGVHD.

Rab13 small G protein and junctional Rab13-binding protein (JRAB) orchestrate actin cytoskeletal organization during epithelial junctional development.

During epithelial junctional development, both vesicle transport and reorganization of the actin cytoskeleton must be spatiotemporally regulated. Coordination of these cellular functions is especially important, but the precise mechanism remains elusive. Previously, we identified junctional Rab13-binding protein (JRAB)/molecules interacting with CasL-like 2 (MICAL-L2) as an effector of the Rab13 small G protein, and we found that the Rab13-JRAB system may be involved in the formation of cell-cell adhesions via transport of adhesion molecules. Here, we showed that JRAB interacts with two actin-binding proteins, actinin-1 and -4, and filamentous actin via different domains and regulates actin cross-linking and stabilization through these interactions. During epithelial junctional development, JRAB is prominently enriched in the actin bundle at the free border; subsequently, JRAB undergoes a Rab13-dependent conformational change that is required for maturation of cell-cell adhesion sites. These results suggest that Rab13 and JRAB regulate reorganization of the actin cytoskeleton throughout epithelial junctional development from establishment to maturation of cell-cell adhesion.

Interaction of nectin-like molecule 2 with integrin alpha6beta4 and inhibition of disassembly of integrin alpha6beta4 from hemidesmosomes.

In normal epithelial cells, integrin α(6)ß(4) is abundantly expressed and forms hemidesmosomes, which is a cellular structure that mediates cell-extracellular matrix binding. In many types of cancer cells, integrin α(6)ß(4) is up-regulated, laminin is cleaved, and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and facilitation of their invasion. We previously showed that the immunoglobulin-like cell adhesion molecule Necl-2 (Nectin-like molecule 2), known as a tumor suppressor, inhibits cancer cell movement by suppressing the ErbB3/ErbB2 signaling. We show here that Necl-2 interacts in cis with integrin α(6)ß(4). The binding of Necl-2 with integrin ß(4) was mediated by its extracellular region. In human colorectal adenocarcinoma Caco-2 cells, integrin α(6)ß(4) was localized at hemidesmosomes. Small interfering RNA-mediated suppression of Necl-2 expression enhanced the phorbol ester-induced disruption of the integrin α(6)ß(4) complex at hemidesmosomes, whereas expression of Necl-2 suppressed the disruption of this structure. These results indicate that tumor-suppressive functions of Necl-2 are mediated by the stabilization of the hemidesmosome structure in addition to the inhibition of the ErbB3/ErbB2 signaling.

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 beta- and alpha-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-DeltaPR1-2, 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.

Interaction of integrin alpha(6)beta(4) with ErbB3 and implication in heregulin-induced ErbB3/ErbB2-mediated DNA synthesis.

Integrin alpha(6)beta(4) is abundantly expressed in normal epithelial cells and forms hemidesmosomes, one of cell-extracellular matrix junctions. In many types of cancer cells, integrin alpha(6)beta(4) is up-regulated, laminin, an integrin alpha(6)beta(4)-binding extracellular matrix protein, is cleaved, and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and a facilitation of their invasion. It was previously shown that integrin alpha(6)beta(4) interacts with ErbB1 and ErbB2 and enhances cell proliferation and motility. Here we show that integrin alpha(6)beta(4) interacts with ErbB3 but not with ErbB1, ErbB2 or ErbB4, and enhances the heregulin-induced, ErbB3/ErbB2 heterodimer-mediated DNA synthesis, but not cell motility, in A549 cells.

Necl-5/PVR enhances PDGF-induced attraction of growing microtubules to the plasma membrane of the leading edge of moving NIH3T3 cells.

Microtubules (MTs) search for and grow toward the leading edge of moving cells, followed by their stabilization at a specific structure at the rear site of the leading edge. This dynamic re-orientation of MTs is critical to directional cell movement. We previously showed that Necl-5/poliovirus receptor (PVR) interacts with platelet-derived growth factor (PDGF) receptor and integrin α(v) ß(3) at the leading edge of moving NIH3T3 cells, resulting in an enhancement of their directional movement. We studied here the role of Necl-5 in the PDGF-induced attraction of growing MTs to the leading edge of NIH3T3 cells. Necl-5 enhanced the PDGF-induced growth of MTs and attracted them near to the plasma membrane of the leading edge of NIH3T3 cells in an integrin α(v) ß(3) -dependent manner. Furthermore, Necl-5 enhanced the PDGF-induced attraction of the plus-end-tracking proteins (+TIPs), including EB1, CLIP170, an intermediate chain subunit of cytoplasmic dynein, and p150(Glued) , a subunit of dynactin, near to the plasma membrane of the leading edge. Thus, Necl-5 plays a role in the attraction of growing MTs to the plasma membrane of the leading edge of moving cells.

The active site structure of nitrided and oxynitrided graphite as a cathode catalyst in a fuel cell.

The first-principles studies were performed for the modified graphites with various edge conformations, which revealed that the NH edge facilitates easy transfer of an electron into the adsorbed O(2).

Inhibition of cell movement and proliferation by cell-cell contact-induced interaction of Necl-5 with nectin-3.

Immunoglobulin-like Necl-5/Tage4/poliovirus receptor (PVR)/CD155, originally identified as the PVR, has been shown to be up-regulated in cancer cells and to enhance growth factor-induced cell movement and proliferation. In addition, Necl-5 heterophilically trans-interacts with nectin-3, a cell-cell adhesion molecule known to form adherens junctions in cooperation with cadherin. We show here that Necl-5 was down-regulated from cell surface upon cell-cell contacts in NIH3T3 cells. This down-regulation of Necl-5 was initiated by its interaction with nectin-3 and was mainly mediated by clathrin-dependent endocytosis. Then, the down-regulation of Necl-5 induced in this way reduced movement and proliferation of NIH3T3 cells. These results indicate that the down-regulation of Necl-5 induced by its interaction with nectin-3 upon cell-cell contacts may be at least one mechanism underlying contact inhibition of cell movement and proliferation.

Novel role of nectin: implication in the co-localization of JAM-A and claudin-1 at the same cell-cell adhesion membrane domain.

Tight junctions (TJs) are formed at the apical side of adherens junctions (AJs) in epithelial cells. Major cell adhesion molecules (CAMs) at TJs are JAM and claudin, whereas major CAMs at AJs are nectin and cadherin. We previously showed that nectin initially forms cell-cell adhesion and then recruits cadherin to the nectin-based cell-cell adhesion sites to form AJs, followed by the recruitment of JAM and claudin to the apical side of AJs to form TJs. We investigated the roles of nectin in the formation of TJs by expressing various combinations of CAMs in L fibroblasts with no TJs or AJs. Co-expression of one of the AJ CAMs and one of the TJ CAMs formed two separate cell-cell adhesion membrane domains (CAMDs). Co-expression of nectin-3 and E-cadherin formed the same CAMD, but co-expression of JAM-A and claudin-1 did not form the same CAMD. Co-expression of JAM-A and claudin-1 with nectin-3, but not E-cadherin, made them form the same CAMD, which was separated from the nectin-based CAMD. Nectin-3 required afadin, a nectin- and F-actin-binding protein, for this ability. In conclusion, nectin plays a novel role in the co-localization of JAM and claudin at the same CAMD.

Establishment of cell polarity by afadin during the formation of embryoid bodies.

Afadin directly links nectin, an immunoglobulin-like cell-cell adhesion molecule, to actin filaments (F-actin) at adherens junctions (AJs). The nectin-afadin complex is important for the formation of not only AJs but also tight junctions (TJs) in epithelial cells. Studies using afadin-knockout mice have revealed that afadin is indispensable for embryonic development by organizing the formation of cell-cell junctions. However, the molecular mechanism of cell-cell junction disorganization during embryonic development in afadin-knockout mice is poorly understood. To address this, we took advantage of embryoid bodies (EBs) as a model system. The formation of cell-cell junctions including AJs and TJs was impaired in afadin-null EBs. The proper accumulation of the Par complex and the activation of Cdc42 and atypical PKC (aPKC), which are crucial for the formation of cell polarity, were also inhibited by knockout of afadin. In addition, the disruption of afadin caused the abnormal deposition of laminin and the dislocalization of its receptors integrin alpha(6) and integrin beta(1). These results indicate that afadin organizes the formation of cell-cell junctions by regulating cell polarization in early embryonic development.