Koji Morimoto

Association of Breast Cancer Stem Cells Identified by Aldehyde Dehydrogenase 1 Expression with Resistance to Sequential Paclitaxel and Epirubicin-Based Chemotherapy for Breast Cancers

Purpose: Breast cancer stem cells have been shown to be associated with resistance to chemotherapy in vitro, but their clinical significance remains to be clarified. The aim of this study was to investigate whether cancer stem cells were clinically significant for resistance to chemotherapy in human breast cancers. Experimental Design: Primary breast cancer patients (n = 108) treated with neoadjuvant chemotherapy consisting of sequential paclitaxel and epirubicin-based chemotherapy were included in the study. Breast cancer stem cells were identified by immunohistochemical staining of CD44/CD24 and aldehyde dehydrogenase 1 (ALDH1) in tumor tissues obtained before and after neoadjuvant chemotherapy. CD44+/CD24− tumor cells or ALDH1-positive tumor cells were considered stem cells. Results: Thirty (27.8%) patients achieved pathologic complete response (pCR). ALDH1-positive tumors were significantly associated with a low pCR rate (9.5% versus 32.2%; P = 0.037), but there was no significant association between CD44+/CD24− tumor cell proportions and pCR rates. Changes in the proportion of CD44+/CD24− or ALDH1-positive tumor cells before and after neoadjuvant chemotherapy were studied in 78 patients who did not achieve pCR. The proportion of ALDH1-positive tumor cells increased significantly (P < 0.001) after neoadjuvant chemotherapy, but that of CD44+/CD24− tumor cells did not. Conclusions: Our findings suggest that breast cancer stem cells identified as ALDH1-positive, but not CD44+/CD24−, play a significant role in resistance to chemotherapy. ALDH1-positive thus seems to be a more significantly predictive marker than CD44+/CD24− for the identification of breast cancer stem cells in terms of resistance to chemotherapy.

CAST: A novel protein of the cytomatrix at the active zone of synapses that forms a ternary complex with RIM1 and Munc13-1

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitter. We have identified here a novel CAZ protein of ∼120 kD from rat brain and named it CAST (CAZ-associated structural protein). CAST had no transmembrane segment, but had four coiled-coil domains and a putative COOH-terminal consensus motif for binding to PDZ domains. CAST was localized at the CAZ of conventional synapses of mouse brain. CAST bound directly RIM1 and indirectly Munc13-1, presumably through RIM1, forming a ternary complex. RIM1 and Munc13-1 are CAZ proteins implicated in Ca2+-dependent exocytosis of neurotansmitters. Bassoon, another CAZ protein, was also associated with this ternary complex. These results suggest that a network of protein–protein interactions among the CAZ proteins exists at the CAZ. At the early stages of synapse formation, CAST was expressed and partly colocalized with bassoon in the axon shaft and the growth cone. The vesicles immunoisolated by antibassoon antibody–coupled beads contained not only bassoon but also CAST and RIM1. These results suggest that these CAZ proteins are at least partly transported on the same vesicles during synapse formation.

Metal-free oxidative cross-coupling of unfunctionalized aromatic compounds.

A new strategy for mixed biaryl synthesis has been developed using the hypervalent iodine(III) reagents. The unique reactivities of the sigma-heteroaryl iodine(III) intermediates generated in situ are the key element for the unusual metal catalyst-free transformations and strict control of the product selectivities.

Stem cell marker aldehyde dehydrogenase 1-positive breast cancers are characterized by negative estrogen receptor, positive human epidermal growth factor receptor type 2, and high Ki67 expression.

Recently, aldehyde dehydrogenase (ALDH) 1 has been identified as a reliable marker for breast cancer stem cells. The aim of our study was to investigate the clinicopathological characteristics of breast cancers with ALDH1+ cancer stem cells. In addition, the distribution of ALDH1+ tumor cells was compared on a cell‐by‐cell basis with that of estrogen receptor (ER)+, Ki67+, or human epidermal growth factor receptor type 2 (HER2)+ tumor cells by means of double immunohistochemical staining. Immunohistochemical staining of ALDH1 was applied to 203 primary breast cancers, and the results were compared with various clinicopathological characteristics of breast cancers including tumor size, histological grade, lymph node metastases, lymphovascular invasion, ER, progesterone receptor, HER2, Ki67, and topoisomerase 2A as well as prognosis. Immunohistochemical double staining of ALDH1 and ER, Ki67, or HER2 was also carried out to investigate their distribution. Of the 203 breast cancers, 21 (10%) were found to be ALDH1+, and these cancers were significantly more likely to be ER− (P = 0.004), progesterone receptor− (P = 0.025), HER2+ (P = 0.001), Ki67+ (P < 0.001), and topoisomerase 2A+ tumors (P = 0.012). Immunohistochemical double staining studies showed that ALDH1+ tumor cells were more likely to be ER−, Ki67−, and HER2+ tumor cells. Patients with ALDH1 (score 3+) tumors showed a tendency (P = 0.056) toward a worse prognosis than did those with ALDH1− tumors. Breast cancers with ALDH1+ cancer stem cells posses biologically aggressive phenotypes that tend to have a poor prognosis, and ALDH1+ cancer stem cells are characterized by ER−, Ki67−, and HER2+. (Cancer Sci 2009; 100: 1062–1068)

Endocytosis of E-cadherin regulated by Rac and Cdc42 small G proteins through IQGAP1 and actin filaments

E-cadherin is a key cell–cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of extracellular signals. To elucidate the mechanism of this endocytosis, we developed here a new cell-free assay system for this reaction using the AJ-enriched fraction from rat liver. We found here that non-trans-interacting, but not trans-interacting, E-cadherin underwent endocytosis in a clathrin-dependent manner. The endocytosis of trans-interacting E-cadherin was inhibited by Rac and Cdc42 small G proteins, which were activated by trans-interacting E-cadherin or trans-interacting nectins, which are known to induce the formation of AJs in cooperation with E-cadherin. This inhibition was mediated by reorganization of the actin cytoskeleton by Rac and Cdc42 through IQGAP1, an actin filament-binding protein and a downstream target of Rac and Cdc42. These results indicate the important role of the Rac/Cdc42-IQGAP1 system in the dynamic organization and maintenance of the E-cadherin–based AJs.

Unusual ipso Substitution of Diaryliodonium Bromides Initiated by a Single‐Electron‐Transfer Oxidizing Process

The diaryliodonium salts ArIArX , which have two aryl groups bound to an iodine atom as a ligand, represent one of the most popular classes of hypervalent iodine compounds and they have application as important arylating agents in organic synthesis. In general, the arylation of nucleophiles with these iodonium salts is assumed to involve the tricoordinated intermediate A before the final ligand-coupling (LC) steps. Owing to the competition of the two LC pathways of Ar and Nu (LCAr ) or Ar and Nu (LCAr ) at the iodine atoms in the intermediates, a mixture of two types of arylated products, Ar Nu and Nu Ar, are potentially obtained in the unsymmetrical salts (Ar1⁄46 Ar) during the aromatic substitution. Previous studies have revealed that the product produced from these pathways should be effected by both electronic and steric factors exerted by the two differential aryl rings of the initial salts—where the nucleophiles would preferentially react with a relatively electron-deficient aryl ring and/or sterically congested ipso carbon atom (i.e. the so-called “ortho effect”). Therefore, the introduction of nucleophiles to an electron-rich heteroaromatic ring is known to be particularly difficult through typical thermal LC processes, which involve the collapse of the intermediate A. Despite their rich chemistry, the utility of diaryliodonium salts as a heteroaryl transfer agent in LC processes has been somewhat limited. Herein, we report a unique single-electron-transfer (SET) oxidizing strategy using diaryliodonium salts as selective heteroaryl transfer agents during ipso substitution. Recently, our research group has reported a metal-free C H coupling method of thiophenes and aromatic compounds using iodonium salts. The method, using TMSBr in hexafluoroisopropanol (HFIP), is useful for the coupling reaction of the thiophene iodonium salts and introduces aromatic nucleophiles to the g positions of the iodine(III)–carbon bonds [Eq. (1)]. However, this reaction is not suitable for coupling through the ipso substitution of the thiophene iodonium salts 1a-X [X = OTs, Br; Eq. (2)].

Roles of cell-adhesion molecules nectin 1 and nectin 3 in ciliary body development

Nectins are Ca2+-independent immunoglobulin-like cell-cell-adhesion molecules consisting of four members. Nectins homophilically and heterophilically trans-interact to form a variety of cell-cell junctions, including cadherin-based adherens junctions in epithelial cells and fibroblasts in culture, synaptic junctions in neurons, and Sertoli cell-spermatid junctions in the testis, in cooperation with, or independently of, cadherins. To further explore the function of nectins, we generated nectin 1–/– and nectin 3–/– mice. Both nectin 1–/– and nectin 3–/– mice showed a virtually identical ocular phenotype, microphthalmia, accompanied by a separation of the apex-apex contact between the pigment and non-pigment cell layers of the ciliary epithelia. Immunofluorescence and immunoelectron microscopy revealed that nectin 1 and nectin 3, but not nectin 2, localized at the apex-apex junctions between the pigment and non-pigment cell layers of the ciliary epithelia. However, nectin 1–/– and nectin 3–/– mice showed no impairment of the apicolateral junctions between the pigment epithelia where nectin 1, nectin 2 and nectin 3 localized, or of the apicolateral junctions between the non-pigment epithelia where nectin 2 and nectin 3, but not nectin 1, localized. These results indicate that the heterophilic trans-interaction between nectin 1 and nectin 3 plays a sentinel role in establishing the apex-apex adhesion between the pigment and non-pigment cell layers of the ciliary epithelia that is essential for the morphogenesis of the ciliary body.

ADIP, a Novel Afadin- and α-Actinin-Binding Protein Localized at Cell-Cell Adherens Junctions

Afadin is an actin filament (F-actin)-binding protein that is associated with the cytoplasmic tail of nectin, a Ca2+-independent immunoglobulin-like cell-cell adhesion molecule. Nectin and afadin strictly localize at cell-cell adherens junctions (AJs) undercoated with F-actin bundles and are involved in the formation of AJs in cooperation with E-cadherin in epithelial cells. In epithelial cells of afadin (−/−) mice and (−/−) embryoid bodies, the proper organization of AJs is markedly impaired. However, the molecular mechanism of how the nectin-afadin system is associated with the E-cadherin-catenin system or functions in the formation of AJs has not yet been fully understood. Here we identified a novel afadin-binding protein, named ADIP (afadin DIL domain-interactingprotein). ADIP consists of 615 amino acids with a calculated M r of 70,954 and has three coiled-coil domains. Northern and Western blot analyses in mouse tissues indicated that ADIP was widely distributed. Immunofluorescence and immunoelectron microscopy revealed that ADIP strictly localized at cell-cell AJs undercoated with F-actin bundles in small intestine absorptive epithelial cells. This localization pattern was the same as those of afadin and nectin. ADIP was undetectable at cell-matrix AJs. ADIP furthermore bound α-actinin, an F-actin-bundling protein known to be indirectly associated with E-cadherin through its direct binding to α-catenin. These results indicate that ADIP is an afadin- and α-actinin-binding protein that localizes at cell-cell AJs and may have two functions. ADIP may connect the nectin-afadin and E-cadherin-catenin systems through α-actinin, and ADIP may be involved in organization of the actin cytoskeleton at AJs through afadin and α-actinin.

JEAP, a Novel Component of Tight Junctions in Exocrine Cells

Tight junctions (TJs) consist of transmembrane proteins and many peripheral membrane proteins. To further characterize the molecular organization of TJs, we attempted here to screen for novel TJ proteins by the fluorescence localization-based expression cloning method. We identified a novel peripheral membrane protein at TJs and named it junction-enriched and -associated protein (JEAP). JEAP consists of 882 amino acids with a calculated molecular weight of 98,444. JEAP contained a polyglutamic acid repeat at the N-terminal region, a coiled-coil domain at the middle region, and a consensus motif for binding to PDZ domains at the C-terminal region. Exogenously expressed JEAP co-localized with ZO-1 and occludin at TJs in polarized Madin-Darby canine kidney cells, but not with claudin-1, JAM, or ZO-1 in L cells. Endogenous JEAP localized at TJs of exocrine cells including pancreas, submandibular gland, lacrimal gland, parotid gland, and sublingual gland, but not at TJs of epithelial cells of small intestine or endothelial cells of blood vessels. The present results indicate that JEAP is a novel component of TJs, which is specifically expressed in exocrine cells.

Metal-Free C–H Cross-Coupling toward Oxygenated Naphthalene-Benzene Linked Biaryls

The intermolecular C-H cross-coupling between aromatic ethers has been achieved for the first time using perfluorinated hypervalent iodine(III) compounds as extreme single-electron-transfer (SET) oxidants. The demonstrations of this specific coupling could provide a direct route to valuable oxygenated mixed naphthalene-benzene biaryls 3 only, without formation of other biaryl-derived byproducts.