Akihiko Ishimura

The Essential Vertebrate ABCE1 Protein Interacts with Eukaryotic Initiation Factors

The ABCE1 gene is a member of the ATP-binding cassette (ABC) multigene family and is composed of two nucleotide binding domains and an N-terminal Fe-S binding domain. The ABCE1 gene encodes a protein originally identified for its inhibition of ribonuclease L, a nuclease induced by interferon in mammalian cells. The protein is also required for the assembly of the HIV and SIV gag polypeptides. However, ABCE1 is one of the most highly conserved proteins and is found in one or two copies in all characterized eukaryotes and archaea. Yeast ABCE1/RLI1 is essential to cell division and interacts with translation initiation factors in the assembly of the pre-initiation complex. We show here that the human ABCE1 protein is essential for in vitro and in vivo translation of mRNA and that it binds to eIF2α and eIF5. Inhibition of the Xenopus ABCE1 arrests growth at the gastrula stage of development, consistent with a block in translation. The human ABCE1 gene contains 16 introns, and the extremely high degree of amino acid identity allows the evolution of its introns to be examined throughout eukaryotes. The demonstration that ABCE1 plays a role in vertebrate translation initiation extends the known functions of this highly conserved protein. Translation is a highly regulated process important to development and pathologies such as cancer, making ABCE1 a potential target for therapeutics. The evolutionary analysis supports a model in which an ancestral eukaryote had large number of introns and that many of these introns were lost in non-vertebrate lineages.

MEG3 Long Noncoding RNA Contributes to the Epigenetic Regulation of Epithelial-Mesenchymal Transition in Lung Cancer Cell Lines.

Histone methylation is implicated in a number of biological and pathological processes, including cancer development. In this study, we investigated the molecular mechanism for the recruitment of Polycomb repressive complex-2 (PRC2) and its accessory component, JARID2, to chromatin, which regulates methylation of lysine 27 of histone H3 (H3K27), during epithelial-mesenchymal transition (EMT) of cancer cells. The expression of MEG3 long noncoding RNA (lncRNA), which could interact with JARID2, was clearly increased during transforming growth factor-β (TGF-β)-induced EMT of human lung cancer cell lines. Knockdown of MEG3 inhibited TGF-β-mediated changes in cell morphology and cell motility characteristic of EMT and counteracted TGF-β-dependent changes in the expression of EMT-related genes such as CDH1, ZEB family, and the microRNA-200 family. Overexpression of MEG3 influenced the expression of these genes and enhanced the effects of TGF-β in their expressions. Chromatin immunoprecipitation (ChIP) revealed that MEG3 regulated the recruitment of JARID2 and EZH2 and histone H3 methylation on the regulatory regions of CDH1 and microRNA-200 family genes for transcriptional repression. RNA immunoprecipitation and chromatin isolation by RNA purification assays indicated that MEG3 could associate with JARID2 and the regulatory regions of target genes to recruit the complex. This study demonstrated a crucial role of MEG3 lncRNA in the epigenetic regulation of the EMT process in lung cancer cells.

Xpbx1b and Xmeis1b play a collaborative role in hindbrain and neural crest gene expression in Xenopus embryos

Pbx1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during embryogenesis and oncogenesis. Pbx proteins bind DNA cooperatively as heterodimers or higher order complexes with Meis family members and Hox proteins and are believed to specify cell identity during development. Here, we present evidence that Pbx1, in partnership with Meis1b, can regulate posterior neural markers and neural crest marker genes during Xenopus development. A Xenopus homolog of the Pbx1b homeodomain protein was isolated and shown to be expressed throughout embryogenesis. Xpbx1b expression overlaps with Xmeis1 in several areas, including the lateral neural folds, caudal branchial arch, hindbrain, and optic cup. When ectopically expressed, Xpbx1b can synergize with Xmeis1b to promote posterior neural and neural crest gene expression in ectodermal explants. Further, a physical interaction between these two homeodomain proteins is necessary for induction of these genes in embryonic tissue. In addition, coexpression of Xmeis1b and Xpbx1b leads to a prominent shift in the localization of Xmeis1b from the cytoplasm to the nucleus, suggesting that nuclear transport or retention of Xmeis1b may depend upon Xpbx1b. Finally, expression of a mutant construct in which Xpbx1b protein is fused to the repressor domain from Drosophila Engrailed inhibits posterior neural and neural crest gene expression. These data indicate that Xpbx1b and its partner, Xmeis1b, function in a transcriptional activation complex during hindbrain and neural crest development.

KDM5B histone demethylase controls epithelial-mesenchymal transition of cancer cells by regulating the expression of the microRNA-200 family.

Histone methylation is implicated in various biological and pathological processes including cancer development. In this study, we discovered that ectopic expression of KDM5B, a histone H3 lysine 4 (H3K4) demethylase, promoted epithelial-mesenchymal transition (EMT) of cancer cells. KDM5B increased the expression of transcription factors, ZEB1 and ZEB2, followed by downregulation of E-cadherin and upregulation of mesenchymal marker genes. The expression of the microRNA-200 (miR-200) family, which specifically targets ZEB1 and ZEB2, was reduced in the cells with KDM5B overexpression. We found that KDM5B repressed the expression of the miR-200 family by changing histone H3 methylation status of their regulatory regions. The introduction of miR-200 precursor in the cells inhibited EMT induction by KDM5B, suggesting that miR-200 family was a critical downstream mediator of KDM5B-promoted EMT. Furthermore, knockdown of KDM5B was shown to affect the expression of EMT-related genes, indicating the involvement of endogenous KDM5B. Our study demonstrated a novel role of KDM5B histone lysine demethylase in EMT, which may contribute to malignant progression of cancer.

JARID2 is involved in transforming growth factor-beta-induced epithelial-mesenchymal transition of lung and colon cancer cell lines.

Histone methylation plays a crucial role in various biological and pathological processes including cancer development. In this study, we discovered that JARID2, an interacting component of Polycomb repressive complex-2 (PRC2) that catalyzes methylation of lysine 27 of histone H3 (H3K27), was involved in Transforming Growth Factor-beta (TGF-ß)-induced epithelial-mesenchymal transition (EMT) of A549 lung cancer cell line and HT29 colon cancer cell line. The expression of JARID2 was increased during TGF-ß-induced EMT of these cell lines and knockdown of JARID2 inhibited TGF-ß-induced morphological conversion of the cells associated with EMT. JARID2 knockdown itself had no effect in the expression of EMT-related genes but antagonized TGF-ß-dependent expression changes of EMT-related genes such as CDH1, ZEB family and microRNA-200 family. Chromatin immunoprecipitation assays showed that JARID2 was implicated in TGF-ß-induced transcriptional repression of CDH1 and microRNA-200 family genes through the regulation of histone H3 methylation and EZH2 occupancies on their regulatory regions. Our study demonstrated a novel role of JARID2 protein, which may control PRC2 recruitment and histone methylation during TGF-ß-induced EMT of lung and colon cancer cell lines.

Tyr-298 in ephrinB1 is critical for an interaction with the Grb4 adaptor protein.

The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, are thought to play a role in the regulation of cell adhesion and migration during development by mediating cell-to-cell signalling events. The transmembrane ephrinB protein is a bidirectional signalling molecule that sends a forward signal through the activation of its cognate receptor tyrosine kinase residing on another cell. The reverse signal is transduced into the ephrinB-expressing cell via tyrosine phosphorylation of its conserved C-terminal cytoplasmic domain. Previous work from our laboratory has implicated the activated FGFR1 (fibroblast growth factor receptor 1) as a regulator of a de-adhesion signal that results from overexpression of ephrinB1. In the present study, we report the isolation of Xenopus Grb4 (growth-factor-receptor-bound protein 4), an ephrinB1-interacting protein, and we show that when expressed in Xenopus oocytes, ephrinB1 interacts with Grb4 in the presence of an activated FGFR1. Amino acid substitutions were generated in Grb4, and the resulting mutants were expressed along with ephrinB1 and an activated FGFR in Xenopus oocytes. Co-immunoprecipitation analysis shows that the FLVR motif within the Src homology 2 domain of Xenopus Grb4 is vital for this phosphorylation-dependent interaction with ephrinB1. More importantly, using deletion and substitution analysis we identify the tyrosine residue at position 298 of ephrinB1 as being required for the physical interaction with Grb4, whereas Tyr-305 and Tyr-310 are dispensable. Moreover, we show that the region between amino acids 301 and 304 of ephrinB1 is also required for this critical tyrosine-phosphorylation-dependent event.

EED regulates epithelial-mesenchymal transition of cancer cells induced by TGF-β.

Histone methylation is involved in various biological and pathological processes including cancer development. In this study, we found that EED, a component of Polycomb repressive complex-2 (PRC2) that catalyzes methylation of lysine 27 of histone H3 (H3K27), was involved in epithelial-mesenchymal transition (EMT) of cancer cells induced by Transforming Growth Factor-beta (TGF-β). The expression of EED was increased during TGF-β-induced EMT and knockdown of EED inhibited TGF-β-induced morphological conversion of the cells associated with EMT. EED knockdown antagonized TGF-β-dependent expression changes of EMT-related genes such as CDH1, ZEB1, ZEB2 and microRNA-200 (miR-200) family. Chromatin immunoprecipitation assays showed that EED was implicated in TGF-β-induced transcriptional repression of CDH1 and miR-200 family genes through the regulation of histone H3 methylation and EZH2 occupancies on their regulatory regions. Our study demonstrated a novel role of EED, which regulates PRC2 activity and histone methylation during TGF-β-induced EMT of cancer cells.

Roles of histone methyl‐modifying enzymes in development and progression of cancer

Retroviral insertional mutagenesis in mice is considered a powerful forward genetic strategy to identify disease genes involved in cancer. Our high‐throughput screens led to frequent identification of the genes encoding the enzymes engaged in histone lysine methylation. Histone methylation can positively or negatively impact on gene transcription, and then fulfill important roles in developmental control and cell‐fate decisions. A tremendous amount of progress has accelerated the characterization of histone methylations and the enzymes that regulate them. Deregulation of these histone methyl‐modifying enzymes has been increasingly recognized as a hallmark of cancer in the last few years. However, in most cases, we have only limited understanding for the molecular mechanisms by which these enzymes contribute to cancer development and progression. In this review, we summarize the current knowledge regarding some of the best‐validated examples of histone lysine methyltransferases and demethylases associated with oncogenesis and discuss their potential mechanisms of action.

DOT1L histone methyltransferase regulates the expression of BCAT1 and is involved in sphere formation and cell migration of breast cancer cell lines

DOT1L is a histone H3 lysine 79 (H3K79) methyltransferase mainly implicated in leukemia. Here we analyzed the function of DOT1L in breast cancer cells. The expression of DOT1L was up-regulated in malignant breast cancer tissues. Over-expression of DOT1L significantly increased the sphere formation and the cell migration activities of MCF7 breast cancer cell line. In contrast, knockdown of DOT1L reduced the cell migration activity of MDA-MB-231 breast cancer cell line. BCAT1, which encodes a branched-chain amino acid transaminase, was identified as one of the target genes controlled by DOT1L through the regulation of H3K79 methylation. Mechanistic investigation revealed that BCAT1 might be an important regulator responsible for DOT1L-mediated sphere formation and cell migration in breast cancer cells.

Oncogenic Met receptor induces cell-cycle progression in Xenopus oocytes independent of direct Grb2 and Shc binding or Mos synthesis, but requires phosphatidylinositol 3-kinase and Raf signaling.

Biological responses of hepatocyte growth factor (HGF) are mediated by the Met receptor tyrosine kinase. Although HGF is a potent mitogen for a variety of cells, the signals required for cell‐cycle progression by the Met/HGF receptor are poorly defined. In this study, we have used the Xenopus oocyte system to define the role of various Met proximal‐binding partners and downstream signaling pathways in cell‐cycle regulation. We show that cell‐cycle progression and activation of MAPK and JNK mediated by the oncogenic Met receptor, Tpr‐Met, are dependent on its kinase activity and the presence of the twin phosphotyrosine (Y482 & Y489) residues in its C‐terminus, but that the recruitment of Grb2 and Shc adaptor proteins is dispensable, implicating other signaling molecules. However, using Met receptor oncoproteins engineered to recruit specific signaling proteins, we demonstrate that recruitment of Grb2 or Shc adaptor proteins is sufficient to induce cell‐cycle progression and activation of MAPK and JNK, while the binding of phospholipase‐Cγ or phosphatidylinositol 3‐kinase alone fails to elicit these responses. Using various means to block phosphatidylinositol 3‐kinase, phospholipase‐Cγ, MEK, JNK, Mos, and Raf1 activity, we show that unlike the fibroblast growth factor receptor, MEK‐dependent and independent signaling contribute to Met receptor‐mediated cell‐cycle progression, but phospholipase‐Cγ or JNK activity and Mos synthesis are not critical. Notably, we demonstrate that Raf1 and phosphatidylinositol 3‐kinase signaling are required for cell‐cycle progression initiated by the Met receptor, a protein frequently deregulated in human tumors. J. Cell. Physiol. 207: 271–285, 2006.